F-box proteins and genes

ABSTRACT

The present invention provides compositions and methods for gene identification, as well as drug discovery and assessment. In particular, the present invention provides components of an E3 complex involved in ubiquitination of cell cycle regulators and other proteins, as well as members of a class of proteins that directly function in recognition of ubiquitination targets. The present invention also provides sequences of multiple F-box proteins.

[0001] This application is a Continuation-in-Part application of U.S.patent application Ser. No. 08/951,621, filed Oct. 16, 1997, pending,which is hereby incorporated herein by reference in its entirety.

[0002] This invention was made with government support under NationalInstitutes of Health Grant No. R01AG11085. The Government has certainrights in the invention.

FIELD OF THE INVENTION

[0003] The present invention provides compositions and methods foridentification of F-box proteins, as well as for drug discovery andassessment. In particular, the present invention provides components ofan E3 complex involved in ubiquitination of cell cycle regulators andother proteins, as well as members of a class of proteins that directlyfunction in recognition of ubiquitination targets.

BACKGROUND OF THE INVENTION

[0004] The proper development and maintenance of a multicellularorganism is a complex process that requires precise spatial and temporalcontrol of cell proliferation. Cell proliferation is controlled via anintricate network of extracellular and intracellular signaling pathwaysthat process growth regulatory signals. This signaling network issuperimposed upon the basic cell cycle regulatory machinery thatcontrols particular cell cycle transitions. In eukaryotes, the cellcycle is comprised of an ordered series of discrete events. In contrastto the periodicity of eukaryotic DNA replication and mitosis, cellulargrowth requires that most metabolic reactions occur continuously. Thecell cycle regulatory machinery coordinates the events that occur duringthe cell cycle, as well as cell growth. Protein degradation is animportant aspect of the development and maintenance of multicellularorganisms, as it provides direction, order, and the appropriate timingfor the key events that occur during the cell cycle.

[0005] The problem of how cell division is controlled has long been atopic of intense research. Early models suggested the existence of aninitiator that would accumulate during the cell cycle, and induce DNAreplication or mitosis when it reached a critical concentration. Themitotic process would then inactivate the initiator, thereby “resetting”the cell cycle. Subsequent research showed that mitotic cyclinsaccumulate during interphase to drive entry of cells into mitosis. Thesecyclins are then degraded at the end of mitosis, in order to reset thecycle. Protein degradation has been shown to have a pervasive role inthe regulation of cell cycle progression. For example, proteolysis isrequired for multiple mitotic processes, and for initiating DNAreplication (See, King et al., Science 274:1652-1659 [1996]).Nonetheless, much remains unknown regarding the proteins and theinteractions that are involved in the proteolytic regulation of the cellcycle and other processes. Indeed, many proteins are likely to beinvolved in proteolysis and cellular maintenance (as well as otherprocesses). Such information is needed for the development of compoundsto regulate the cell cycle and prevent or treat diseases associated withabnormal cell proliferation.

SUMMARY OF THE INVENTION

[0006] The present invention provides compositions and methods for geneidentification (e.g., F-box genes), as well as drug discovery andassessment. The present invention provides components of an E3 complexinvolved in ubiquitination of cell cycle regulators and other proteins,as well as members of a class of proteins that directly function inrecognition of ubiquitination targets.

[0007] Thus, the present invention provides the function of a class ofproteins referred to as F-box proteins in targeted ubiquitination. Thepresent invention finds utility in methods for developing compounds thataffect ubiquitination. The present invention also provides numerousnovel F-box containing mammalian genes whose encoded proteins arecontemplated to function in processes including, but not limited, totargeted ubiquitination of cellular proteins.

[0008] The present invention also provides amino acid and DNA sequenceinformation for eighteen novel F-box-containing human or mouse genes. Aswith Cdc4, Grr1, Skp2, and cyclin F, these novel F-box proteins have thecapacity to associate with Skp1 and to simultaneously interact withother proteins through other protein-protein interaction motifs encodedby regions of their genes other than the F-box. Thus, the presentinvention provides compositions and methods for determining theinteraction of these proteins with other proteins.

[0009] In one embodiment, the present invention provides an isolatedpolypeptide comprising at least one functionally active fragment of anF-box protein. In a preferred alternative embodiment, the F-box proteinis mammalian, while in a particularly preferred embodiment, the F-boxprotein is human or murine.

[0010] In another embodiment, the functionally active fragment comprisesthe amino acid sequence selected from the amino acid sequences set forthin SEQ ID NOS:1, 3, 5, 9, 13, 17, 19, 25, 27, 41, 45, 47, 51, 53, 55,and 57, while in alternative embodiment, the functionally activefragment comprises the amino acid sequence selected from the amino acidsequences set forth in SEQ ID NOS:7, 11, 15, 21, 23, 29, 31, 33, 35, 37,39, 43, and 49.

[0011] The present invention also provides a purified antibody whichbinds specifically to the isolated polypeptide encoding an F-boxprotein. In one embodiment, the antibody is monoclonal, while in anotherembodiment, the antibody is polyclonal. In another embodiment, thepresent invention provides a purified antibody which specifically bindsto a complex comprised of an F-box protein and an F-box protein target.In yet another embodiment, the present invention provides an antibodywhich specifically binds to a complex comprised of an F-box protein andSkp1; it is contemplated that the Skp1 in the complex may be bound toanother protein, but such binding is not required.

[0012] The present invention also provides an isolated nucleotidesequence encoding at least one functionally active fragment of an F-boxprotein, wherein the nucleotide sequence encodes at least a portion ofan F-box protein. In a preferred embodiment, the F-box protein ismammalian, while in particularly preferred embodiments, the F-boxprotein is human or murine. In one embodiment, the isolated nucleotidesequence comprises at least a portion of the sequence set forth in SEQID NOS:2, 4, 6, 10, 14, 18, 20, 26, 28, 42, 48, 52, 54, 56, and 58. Inanother embodiment, the isolated nucleotide sequence comprises at leasta portion of the sequence set forth in SEQ ID NO:8, 12, 16, 22, 24, 30,32, 34, 36, 38, 40, 44, and 50.

[0013] The present invention also provides a vector comprising anucleotide sequence, wherein the nucleotide sequence comprises thenucleotide sequence encoding at least one functionally active fragmentof an F-box protein, wherein the nucleotide sequence encodes at least aportion of an F-box protein. In one preferred embodiment, the isolatednucleotide sequence comprises at least a portion of the sequence setforth in SEQ ID NOS:2, 4, 6, 10, 14, 18, 20, 26, 28, 42, 48, 52, 54, 56,and 58, while in another preferred embodiment, the isolated nucleotidesequence comprises at least a portion of the sequence set forth in SEQID NO:8, 12, 16, 22, 24, 30, 32, 34, 36, 38, 40, 44, and 50.

[0014] The present invention also provides a host cell transformed withat least one vector comprising a nucleotide sequence, wherein thenucleotide sequence comprises the nucleotide sequence encoding at leastone functionally active fragment of an F-box protein, wherein thenucleotide sequence encodes at least a portion of an F-box protein. Inone preferred embodiment, the isolated nucleotide sequence comprises atleast a portion of the sequence set forth in SEQ ID NOS:2, 4, 6, 10, 14,18, 20, 26, 28, 42, 48, 52, 54, 56, and 58, while in another preferredembodiment, the isolated nucleotide sequence comprises at least aportion of the sequence set forth in SEQ ID NO:8, 12, 16, 22, 24, 30,32, 34, 36, 38, 40, 44, and 50.

[0015] The present invention also provides an isolated nucleotidesequence encoding the amino acid sequence selected from group consistingof SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, 15 ,17, 19, 21, 23, 25, 27, 29, 31,33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, and 57. In oneembodiment, the present invention provides a vector comprising anisolated nucleotide sequence encoding the amino acid sequence selectedfrom group consisting of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13,15 ,17, 19,21, 23, 25, 27, 29,31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55,and 57. In another embodiment, the present invention provides a hostcell transformed with this vector.

[0016] The present invention further provides a polynucleotide sequencecomprising at least fifteen nucleotides, which hybridizes understringent conditions to at least a portion of a polynucleotide sequence,wherein the polynucleotide sequence is selected from the polynucleotidesequences set forth in SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 18, 20,22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56,and 58.

[0017] The present invention also provides methods for detection ofpolynucleotides encoding F-box protein in a biological sample comprisingthe steps of: hybridizing at least a portion of the polynucleotideencoding an F-box protein, to nucleic acid material of a biologicalsample, thereby forming a hybridization complex; and detecting thehybridization complex, wherein the presence of the complex correlateswith the presence of a polynucleotide encoding F-box protein in thebiological sample. In one embodiment of the method, prior tohybridization, the nucleic acid material of the biological sample isamplified by the polymerase chain reaction.

[0018] The present invention also provides methods for the detection ofF-box protein targets comprising the steps of: providing an F-boxprotein, and a sample suspected of containing an F-box protein target;exposing the F-box protein to the sample under conditions such that theF-box protein binds to the F-box protein target to form an F-box proteinand target complex; and detecting the F-box protein and target complex.In one embodiment of the method, the box protein target is selected fromthe group consisting of cyclins, cyclin-dependent kinases, and IκB. Analternative embodiment further comprises the step of analyzing saidF-box protein and target complex, wherein the analyzing comprisesobserving the F-box protein and target complex for degradation of theF-box protein target. In another embodiment, the method furthercomprises the step of exposing the F-box protein and F-box proteintarget to an F-box protein antagonist. In yet another embodiment of themethod, the F-box protein antagonist prevents the formation of the F-boxprotein and the target complex.

[0019] The present invention also provides methods for the detection ofan F-box protein and Skp1 complex, comprising the steps of: providing anF-box protein, and Skp1; exposing the F-box protein to Skp1 underconditions such that the F-box protein binds to Skp1 to form an F-boxprotein and Skp1 complex; and detecting the F-box protein and Skp1complex. One embodiment of the method further comprises the step ofexposing the F-box protein and Skp1 to an F-box protein antagonist. Inyet another embodiment of the method, the F-box protein antagonistprevents the formation of the F-box protein and Skp1 complex.

[0020] The present invention also provides methods and compositionsuseful to determine the complexity and diversity of mammalian F-boxproteins, as well as the identity of F-box proteins from variousspecies, the protein-protein interaction domains involved, theproteolytic pathways, and regulatory pathways. Indeed, the presentinvention provides methods and compositions to identify the functionsand ubiquitination targets of these and other F-box containing proteins.

[0021] However, the present invention is not limited to F-box proteinsinvolved in ubiquitination. Thus, the function of F-box proteins is notnecessarily limited to ubiquitination, and the present inventionprovides the methods and compositions to make this determination. It iscontemplated that additional F-box containing genes will be discoveredthrough the use of two-hybrid screens with Skp1 or ubiquitinationtargets as the two-hybrid “bait” (e.g., as described in the Example 6).It is also contemplated that additional F-box genes will be discoveredthrough sequencing of the mammalian genome and sequence analysis, todetermine the homology with existing F-box proteins, such as thoseidentified in the present invention.

[0022] The present invention also provide compositions and methods fordevelopment of drugs that disrupt at least one pathway in which F-boxproteins function, and are required for biological and/or biochemicalprocesses.

[0023] The present invention also provides methods and compositions toidentify and/or investigate cell cycle regulators, transcriptionregulators, proteins involved in DNA replication, and other cellularregulatory proteins. It is further contemplated that the presentinvention finds use in elucidating inflammatory response and infectiousdisease processes involving protein degradation, as well as developmentof compounds that control (i.e., either enhance or retard) proteindegradation, as appropriate to ameliorate the effects of theinflammatory response or disease process.

[0024] The present invention also provides methods and compositions foridentifying and investigating the function of protein targets whoseabundance is altered in disease, as well as for detection,identification, and characterization of mutations in F-box genes throughvarious methods, including, but not limited sequence analysis, Southernblot analysis of DNA, etc. Furthermore, the present invention also findsuse in assessing alterations in cellular protein abundance due tooverexpression of particular F-box proteins. It is contemplated thatsuch alterations are associated with particular diseases. The presentinvention also finds use in determination of overexpression caused bygene amplification in DNA samples from diseased tissue or individualsthrough such methods as Southern analysis using a particular F-box geneas probe.

[0025] It is also contemplated that targets of novel human F-boxproteins will be determined by those experienced in the art byapproaches including, but not limited to two-hybrid library screens,immunoprecipitation analysis followed by immunoblotting with antibodiesagainst candidate targets, peptide mapping, mass spectral analysis,peptide sequencing, and/or by screening lambda based expressionlibraries with F-box protein probes.

[0026] In addition, the present invention finds use in engineering F-boxproteins to artificially recruit particular proteins into an E3 complexfor ubiquitination. Thus, it is clear that the present inventionprovides methods and compositions for detailed investigation of F-boxproteins, as well as proteins that associate with F-box proteins.Furthermore, the present invention thereby provides methods andcompositions for the detection and analysis of abnormalities inproteolytic functions, as well as methods and compositions for thedevelopment of compounds suitable for use in ameliorating suchabnormalities.

[0027] The present invention further provides a method for the detectionof one or more NF-κB regulatory factors comprising the steps of:providing a slimb protein, and a sample suspected of containing one ormore NF-κB regulatory factors, and exposing the slimb protein to thesample under conditions such that the slimb protein binds to the one ormore NF-κB regulatory factors to form a slimb/regulatory factor complex.In some preferred embodiments, the method further comprises the step ofdetecting the slimb/regulatory factor complex. In other embodiment, themethod further comprises the step of observing the slimb/regulatoryfactor complex for degradation of the one or more NF-κB regulatoryfactors. In yet other embodiments, the method further comprises the stepof exposing the slimb protein and one or more NF-κB regulatory factorsto an F-box protein antagonist. In particular embodiments, the F-boxprotein antagonist prevents the formation of the slimb/regulatory factorcomplex.

[0028] The present invention also provides a method for the detection ofa slimb protein complex, comprising the steps of: providing a slimbprotein and a sample suspected of containing one or more proteinscapable of forming a complex with the slimb protein; and exposing theslimb protein to the one or more proteins capable of forming a complexwith the slimb protein under conditions such that the slimb proteinbinds to the one or more proteins capable of forming a complex with theslimb protein to form a slimb protein complex. In preferred embodiments,the method further comprises the step of detecting the slimb proteincomplex. In some embodiment, step b) of the method further comprisesexposing the slimb protein and the one or more proteins capable offorming a complex with the slimb protein to an F-box protein antagonist.In particularly preferred embodiments, the F-box protein antagonistprevents the formation of the slimb protein complex.

DESCRIPTION OF THE FIGURES

[0029] Unless otherwise indicated, a “P” enclosed within a circleindicates that the protein associated with the symbol is phosphorylated.

[0030]FIG. 1 shows the assembly of a multiprotein complex containingCdc34, Cdc53, Skp1, and Cdc4, with the three panels showing theenhancement of the formation of a Cdc53/Cdc4 complex by Skp1. Panel Ashows the results of immunoprecipitation with Myc tag on Cdc53(Cdc53^(M)) using anti-Myc antibodies. Panel B shows theimmunoprecipitation results with a Flag tag on Cdc4 (Cdc4^(F)). Panel Cshows that Skp1 and Cdc34 can associate with Cdc53 simultaneously.

[0031]FIG. 2A shows an SDS-PAGE analysis of purified Cln1HA/Gst-Cdc28HA/Cks1.

[0032]FIG. 2B is an autoradiograph showing the phosphorylation of Sic1by Cln1/Cdc28 complexes in vitro.

[0033]FIG. 2C shows immunoblot results indicating that phosphorylationof Sic1 is required for its association with Cdc34/Cdc53/Skp1/Cdc4complexes.

[0034]FIG. 2D shows immunoblot results indicating that association ofphosphorylated Sic1 with Cdc4 is enhanced by Skp1.

[0035]FIG. 2E shows immunoblot results indicating that association ofphosphorylated Sic1 with Skp1 requires the WD-40 repeats of Cdc4.

[0036]FIG. 3A shows the immunoblot results demonstrating thatphosphorylated Sic1 is ubiquitinated in vivo and in vitro with purifiedCdc34 E2 and Cdc53/Skp1/Cdc4 complexes.

[0037]FIG. 3B shows immunoblot results for anti-Cdc53^(M) immunecomplexes tested against Cdc53^(M)/Skp1, Cdc53^(M)/Skp1/Cdc4, andsupplemented with ATP, ubiquitin, human E1, Cdc34 purified from E. coli,and either unphosphorylated or phosphorylated Sic1 complexes.

[0038]FIG. 3C shows immunoblot results for anti-Skp1^(F) immunecomplexes tested with SkP1^(F)/Cdc53^(M)/Cdc4, Skp1^(F)/Cdc4, andSkp1^(F)/Cdc53^(M).

[0039]FIG. 3D shows immunoblot results that indicate ubiquitination ofSic1 does not require that Cln/Cdc28 be present in the ubiquitinationreaction nor that Sic1 be associated with Clb5/Cdc28.

[0040]FIG. 3E shows immunoblot results that Clb5/Cdc28-phosphorylatedSic1 is a substrate for ubiquitination by Cdc34.

[0041]FIG. 4A shows immunoblot results indicating that Grr1 canassociate with Skp1 and Cdc53.

[0042]FIG. 4B shows an autoradiograph indicating that phosphorylatedSic1 associates with Cdc4 but not Grr1-containing complexes.

[0043]FIG. 4C shows an immunoblot indicating that Cdc4, but not Grr1,supports ubiquitination of Sic1 in vitro.

[0044]FIG. 4D shows an immunoblot used to verify the presence ofreaction components derived from immunoprecipitation (the blot used forubiquitination assays was reprobed to detect Grr1G10, Cdc53^(M), andCdc4).

[0045]FIG. 5A is an autoradiograph showing differential recognition ofSic1 and Cln proteins by Grr1 and Cdc4.

[0046]FIG. 5B shows an immunoblot verifying the presence of Cdc4,Grr1G10, Cdc53^(M), and Skp1^(F).

[0047]FIG. 5C is an immunoblot indicating that phosphorylation of Cln isrequired for the association of Cln1/Cdc28 complexes with Grr1.

[0048]FIG. 5D is an autoradiograph showing that purified Skp1/Cdc53/Grr1complexes are not sufficient for Cln1 ubiquitination by Cdc34 in vitro.

[0049]FIG. 5E is an immunoblot showing that phosphorylated Cln1 isubiquitinated in a fractionated yeast extract system.

[0050]FIG. 6A is a schematic showing that phosphorylation of substratesthrough protein kinase signalling pathways is required for recognitionby F-box receptor proteins.

[0051]FIG. 6B is a schematic showing that distinct F-box complexes mayregulate different biological processes through selective recruitment ofsubstrates. Hypothetical FEC configurations are shown together with thesignals that are being sensed, the corresponding substrates and thephysiological consequences of complex function.

[0052]FIG. 6C is a schematic showing the interplay between proteinkinase and the SCF pathway in the G1 to S-phase transition in S.cerevisiae. In this Figure, perpendicular bars indicate inhibitoryevents.

[0053]FIG. 7 shows the alignment of various F-box proteins provided inthe present invention.

[0054]FIG. 8 shows immunoblotting results demonstrating that Skp1associates with phosphorylated IκB but not unphosphorylated IκB.

[0055]FIG. 9 shows the interaction between various F-box proteins andphosphorylated and unphosphorylated IκB. Schematic representations ofthe F-box proteins are provided with “F” representing F-box sequences.

[0056]FIGS. 10A and 10B show lysates from the indicated transfectionsthat were subjected to immunoblotting using the indicated antibodies.

[0057]FIGS. 11A and 11B show additional immunoprecipitation/westernblotting experiments using the indicated transfections and antibodies.

[0058]FIG. 12 shows immunoblotting results with the indicated antibodiesfor phosphorylation-specific interaction of SCF slimb complexes with IκBpeptide sequences.

DEFINITIONS

[0059] To facilitate understanding of the invention, a number of termsare defined below.

[0060] As used herein, the term “F-box proteins” refers to the aminoacid sequences of substantially purified proteins involved inproteolysis, including but not limited to proteins involved in theubiquitin-ligase complex obtained from any species, including bovine,ovine, porcine, murine, equine, and human, from any source whethernatural, synthetic, semi-synthetic, or recombinant. The F-box is asequence of 35-45 amino acids and allows the F-box proteins to enterinto complexes with Skp1. Thus, the F-box proteins bind Skp1, andcontain a motif that displays sequence similarity to Grr1 and Cdc4. Thisconserved structural motif is included in the sequence alignments shownin FIG. 7 (i.e., the amino acid residues that are shared by the F-boxproteins shown). However, it is not intended that the term be limited tothe exact sequences set forth in FIG. 7. In some embodiments, the F-boxproteins further comprise additional motifs, in particular motifsinvolved in protein-protein interaction. These additional motifsincluded, but are not limited to leucine-rich repeats, and WD-40. Inpreferred embodiments, the F-box protein is mammalian, while inparticularly preferred embodiments, the F-box protein is human ormurine.

[0061] As used herein, the term “F-box target” refers to any moiety thatis recognized by at least one F-box containing protein. It is intendedthat the term encompass such proteins as the cyclins (e.g., A, D, andE), as well as cyclin kinase inhibitors (e.g., p27), and IκB, as well asother proteins. It is not intended that the term be limited to anyparticular protein or compound.

[0062] As used herein, the term “multiprotein complex” refers tocomplexes comprising more than one protein. It is intended that the termencompass complexes with any number of proteins. In preferredembodiments, the proteins comprising a multiprotein complex functioncooperatively. For example, in particularly preferred embodiments of thepresent invention, Cdc34, Cdc53, Skp1, and Cdc4 comprise a multiproteincomplex. It is also intended that the term encompass complexescomprising Skp1, any of the amino acid sequences set forth in Table 2 orTable 4, and a Cdc53 homolog. In preferred embodiments, the Cdc53homolog in such multiprotein complexes comprises human Cul proteins(e.g., Cul 1 through 5), as well as murine Cul proteins. It is alsointended that this term encompass complexes comprised of an F-boxprotein and its target protein (i.e., an F-box target protein).

[0063] The term “modulate,” as used herein, refers to a change or analteration in the biological activity of an F-box protein (e.g.,mammalian F-box proteins). Modulation may be an increase or a decreasein protein activity, a change in binding characteristics, or any otherchange in the biological, functional, or immunological properties of anF-box protein.

[0064] The term “mimetic,” as used herein, refers to a molecule, thestructure of which is developed from knowledge of the structure of anF-box protein, or portions thereof and, as such, is able to effect someor all of the actions of F-box proteins and/or F-box protein-likemolecules.

[0065] The term “antagonist” refers to molecules or compounds whichinhibit the action of a composition (e.g., an F-box protein).Antagonists may or may not be homologous to the targets of thesecompositions in respect to conformation, charge or othercharacteristics. In particularly preferred embodiments, antagonistsprevent the functioning of F-box proteins. It is contemplated thatantagonists may prevent binding of an F-box protein and its target(s).It is also contemplated that antagonists prevent or alter the binding ofan F-box protein and Skp1. However, it is not intended that the term belimited to a particular site of function.

[0066] The term “derivative,” as used herein, refers to the chemicalmodification of a nucleic acid encoding an F-box protein (in particular,mammalian F-box proteins), or the encoded F-box protein. Illustrative ofsuch modifications would be replacement of hydrogen by an alkyl, acyl,or amino group. A nucleic acid derivative would encode a polypeptidewhich retains essential biological characteristics of the naturalmolecule.

[0067] A “variant” of an F-box protein, as used herein, refers to anamino acid sequence that is altered by one or more amino acids. Thevariant may have “conservative” changes, wherein a substituted aminoacid has similar structural or chemical properties (e.g., replacement ofleucine with isoleucine). More rarely, a variant may have“nonconservative” changes (e.g., replacement of a glycine with atryptophan). Similar minor variations may also include amino aciddeletions or insertions, or both. Guidance in determining which aminoacid residues may be substituted, inserted, or deleted withoutabolishing biological or immunological activity may be found usingcomputer programs well known in the art, for example, DNASTAR software.

[0068] “Alterations” in the polynucleotide of for example, SEQ ID NO:4,as used herein, comprise any alteration in the sequence ofpolynucleotides encoding human F1 Alpha F-box protein, includingdeletions, insertions, and point mutations that may be detected usinghybridization assays. Included within this definition is the detectionof alterations to the genomic DNA sequence which encodes an F-boxprotein (e.g., by alterations in the pattern of restriction fragmentlength polymorphisms) capable of hybridizing to a particular sequence,the inability of a selected fragment to hybridize to a sample of genomicDNA (e.g., using allele-specific oligonucleotide probes), and improperor unexpected hybridization, such as hybridization to a locus other thanthe normal chromosomal locus for the polynucleotide sequence encoding anF-box protein (e.g., using fluorescent in situ hybridization [FISH] tometaphase chromosomes spreads).

[0069] A “consensus gene sequence” refers to a gene sequence which isderived by comparison of two or more gene sequences and which describesthe nucleotides most often present in a given segment of the genes; theconsensus sequence is the canonical sequence. In some embodiments,“consensus,” refers to a nucleic acid sequence which has beenresequenced to resolve uncalled bases, or which has been extended usingany suitable method known in the art, in the 5′ and/or the 3′ directionand resequenced, or which has been assembled from the overlappingsequences of more than one clone using any suitable method known in theart, or which has been both extended and assembled.

[0070] The term “sample,” as used herein, is used in its broadest sense.The term encompasses biological sample(s) suspected of containingnucleic acid encoding F-box proteins or fragments thereof, and maycomprise a cell, chromosomes isolated from a cell (e.g., a spread ofmetaphase chromosomes), genomic DNA (in solution or bound to a solidsupport such as for Southern analysis), RNA (in solution or bound to asolid support such as for northern analysis), cDNA (in solution or boundto a solid support), an extract from cells or a tissue, and the like.

[0071] As used herein the terms “protein” and “polypeptide” refer tocompounds comprising amino acids joined via peptide bonds and are usedinterchangeably.

[0072] The terms “gene sequences” or “native gene sequences” are used toindicate DNA sequences encoding a particular gene which contain the sameDNA sequences as found in the gene as isolated from nature. In contrast,“synthetic gene sequences” are DNA sequences which are used to replacethe naturally occurring DNA sequences when the naturally occurringsequences cause expression problems in a given host cell. For example,naturally-occurring DNA sequences encoding codons which are rarely usedin a host cell may be replaced (e.g., by site-directed mutagenesis) suchthat the synthetic DNA sequence represents a more frequently used codon.The native DNA sequence and the synthetic DNA sequence will preferablyencode the same amino acid sequence.

[0073] As used herein, the term “gene” means the deoxyribonucleotidesequences comprising the coding region of a structural gene and theincluding sequences located adjacent to the coding region on both the 5,and 3, ends for a distance of about 1 kb on either end such that thegene corresponds to the length of the full-length mRNA. The sequenceswhich are located 5′ of the coding region and which are present on themRNA are referred to as 5′ non-translated sequences. The sequences whichare located 3′ or downstream of the coding region and which are presenton the mRNA are referred to as 3′ non-translated sequences; thesesequences. The term “gene” encompasses both cDNA and genomic forms of agene. A genomic form or clone of a gene contains the coding regioninterrupted with non-coding sequences termed “introns” or “interveningregions” or “intervening sequences.” Introns are segments of a genewhich are transcribed into nuclear RNA (hnRNA); introns may containregulatory elements such as enhancers. Introns are removed or “splicedout” from the nuclear or primary transcript; introns therefore areabsent in the messenger RNA (mRNA) transcript. The mRNA functions duringtranslation to specify the sequence or order of amino acids in a nascentpolypeptide.

[0074] In addition to containing introns, genomic forms of a gene mayalso include sequences located on both the 5′ and 3′ end of thesequences which are present on the RNA transcript. These sequences arereferred to as “flanking” sequences or regions (these flanking sequencesare located 5′ or 3′ to the non-translated sequences present on the mRNAtranscript). The 5′ flanking region may contain regulatory sequencessuch as promoters and enhancers which control or influence thetranscription of the gene. The 3′ flanking region may contain sequenceswhich direct the termination of transcription, post-transcriptionalcleavage and polyadenylation.

[0075] As used herein, the term “structural gene” refers to a DNAsequence coding for RNA or a protein. In contrast, “regulatory genes”are structural genes which encode products which control the expressionof other genes (e.g., transcription factors).

[0076] As used herein the term “coding region” when used in reference tostructural gene refers to the nucleotide sequences which encode theamino acids found in the nascent polypeptide as a result of translationof a mRNA molecule. The coding region is bounded, in eukaryotes, on the5′ side by the nucleotide triplet “ATG” which encodes the initiatormethionine and on the 3′ side by one of the three triplets which specifystop codons (i.e., TAA, TAG, TGA).

[0077] The term “portion,” as used herein, with regard to a protein (asin “a portion of a given protein”) refers to fragments of that protein.The fragments may range in size from four amino acid residues to theentire amino acid sequence minus one amino acid. Thus, a protein“comprising at least a portion of the amino acid sequence of SEQ IDNO:3” encompasses the full-length human F1 protein, and fragmentsthereof.

[0078] “Nucleic acid sequence” as used herein refers to anoligonucleotide, nucleotide, or polynucleotide, and fragments orportions thereof, and to DNA or RNA of genomic or synthetic origin whichmay be single- or double-stranded, and represent the sense or antisensestrand. Similarly, “amino acid sequence” as used herein refers to anoligopeptide, peptide, polypeptide, or protein sequence, and fragmentsor portions thereof, and to naturally occurring or synthetic molecules.

[0079] A “composition comprising a given polynucleotide sequence” asused herein refers broadly to any composition containing the givenpolynucleotide sequence. The composition may comprise an aqueoussolution. Compositions comprising polynucleotide sequences encodingF-box proteins or fragments thereof, may be employed as hybridizationprobes. In this case, the F-box-encoding polynucleotide sequences aretypically employed in an aqueous solution containing salts (e.g., NaCl),detergents (e.g., SDS) and other components (e.g., Denhardt's solution,dry milk, salmon sperm DNA, etc.).

[0080] Where “amino acid sequence” is recited herein to refer to anamino acid sequence of a naturally occurring protein molecule, “aminoacid sequence” and like terms, such as “polypeptide” or “protein” arenot meant to limit the amino acid sequence to the complete, native aminoacid sequence associated with the recited protein molecule.

[0081] A “deletion,” as used herein, refers to a change in either aminoacid or nucleotide sequence in which one or more amino acid ornucleotide residues, respectively, are absent.

[0082] An “insertion” or “addition,” as used herein, refers to a changein an amino acid or nucleotide sequence resulting in the addition of oneor more amino acid or nucleotide residues, respectively, as compared tothe naturally occurring molecule.

[0083] A “substitution,” as used herein, refers to the replacement ofone or more amino acids or nucleotides by different amino acids ornucleotides, respectively.

[0084] The term “biologically active,” as used herein, refers to aprotein having structural, regulatory, or biochemical functions of anaturally occurring molecule. Likewise, “immunologically active” refersto the capability of the natural, recombinant, or synthetic F-boxproteins, or any oligopeptide thereof, to induce a specific immuneresponse in appropriate animals or cells and to bind with specificantibodies.

[0085] As used herein, the term “purified” or “to purify” refers to theremoval of contaminants from a sample. For example, proteins of interestare purified by removal of contaminating proteins; they are alsopurified by the removal of substantially all proteins that are not ofinterest. The removal of non-immunoglobulin proteins and/or the removalof immunoglobulins that do not bind protein results in an increase inthe percent of protein of interest-reactive immunoglobulins in thesample. In another example, recombinant polypeptides are expressed inbacterial host cells and the polypeptides are purified by the removal ofhost cell proteins; the percent of recombinant polypeptides is therebyincreased in the sample.

[0086] The term “substantially purified,” as used herein, refers tonucleic or amino acid sequences that are removed from their naturalenvironment, isolated or separated, and are at least 60% free,preferably 75% free, and most preferably 90% free from other componentswith which they are naturally associated.

[0087] The term “recombinant DNA molecule” as used herein refers to aDNA molecule which is comprised of segments of DNA joined together bymeans of molecular biological techniques.

[0088] The term “recombinant protein” or “recombinant polypeptide” asused herein refers to a protein molecule which is expressed from arecombinant DNA molecule. The term “native protein” as used hereinrefers to a protein which is isolated from a natural source as opposedto the production of a protein by recombinant means.

[0089] As used herein, the term “overproducing” is used in reference tothe production of polypeptides in a host cell, and indicates that thehost cell is producing more of the polypeptide by virtue of theintroduction of nucleic acid sequences encoding the polypeptide thanwould be expressed by the host cell absent the introduction of thesenucleic acid sequences. To allow ease of purification of polypeptidesproduced in a host cell it is preferred that the host cell express oroverproduce the polypeptide at a level greater than 1 mg/liter of hostcell culture.

[0090] “A host cell capable of expressing a recombinant protein as asoluble protein at a level greater than or equal to X milligrams per 1OD of cells per liter” is a host cell that produces X milligrams ofrecombinant protein per liter of culture medium containing a density ofhost cells equal to 1 OD₆₀₀. The amount of recombinant protein presentper OD per liter is determined by quantitating the amount of recombinantprotein recovered following affinity purification.

[0091] “A host cell capable of secreting a recombinant protein into theculture supernatant at a level greater than or equal to 10 mgrecombinant protein per 1 OD of cells per liter” refers to a host cellthat secretes a recombinant protein into the culture supernatant (i.e.,the medium, such as LB broth, used to grow the host cell) at a levelgreater than or equal to 10 mg recombinant protein per liter of mediumcontaining a concentration (i.e., density) of host cells equal to 1OD₆₀₀. The host cells may be grown in shaker flasks (approximately 1liter culture medium) or in fermentation tank (approximately 10 litersculture medium) and the amount of recombinant protein secreted into theculture supernatant may be determined using a quantitative ELISA assay.

[0092] As used herein, the term “fusion protein” refers to a chimericprotein containing the protein of interest (i.e., a ubiquitinationcomplex and/or fragments thereof) joined to an exogenous proteinfragment (the fusion partner which consists of a non-ubiquitinationcomplex protein). The fusion partner may enhance solubility of theprotein as expressed in a host cell, may provide an “affinity tag” toallow purification of the recombinant fusion protein from the host cellor culture supernatant, or both. If desired, the fusion protein may beremoved from the protein of interest prior to immunization by a varietyof enzymatic or chemical means known to the art.

[0093] As used herein, the term “affinity tag” refers to such structuresas a “poly-histidine tract” or “poly-histidine tag,” or any otherstructure or compound which facilitates the purification of arecombinant fusion protein from a host cell, host cell culturesupernatant, or both. As used herein, the term “flag tag” refers toshort polypeptide marker sequence useful for recombinant proteinidentification and purification.

[0094] As used herein, the terms “poly-histidine tract” and“poly-histidine tag,” when used in reference to a fusion protein refersto the presence of two to ten histidine (or more) residues at either theamino- or carboxy-terminus of a protein of interest. A poly-histidinetract of six to ten residues is preferred. The poly-histidine tract isalso defined functionally as being a number of consecutive histidineresidues added to the protein of interest which allows the affinitypurification of the resulting fusion protein on a nickel-chelate or IDAcolumn.

[0095] As used herein, the term “chimeric protein” refers to two or morecoding sequences obtained from different genes, that have been clonedtogether and that, after translation, act as a single polypeptidesequence. Chimeric proteins are also referred to as “hybrid proteins.”As used herein, the term “chimeric protein” refers to coding sequencesthat are obtained from different species of organisms, as well as codingsequences that are obtained from the same species of organisms.

[0096] As used herein, the term “protein of interest” refers to theprotein whose expression is desired within the fusion protein. In afusion protein, the protein of interest will be joined or fused withanother protein or protein domain, the fusion partner, to allow forenhanced stability of the protein of interest and/or ease ofpurification of the fusion protein.

[0097] As used herein “soluble” when in reference to a protein producedby recombinant DNA technology in a host cell, is a protein which existsin solution in the cytoplasm of the host cell; if the protein contains asignal sequence, the soluble protein is secreted into the culture mediumof eukaryotic cells capable of secretion or by bacterial hostspossessing the appropriate genes. In contrast, an insoluble protein isone which exists in denatured form inside cytoplasmic granules (i.e.,inclusion bodies) in the host cell. High level expression (i.e., greaterthan 1 mg recombinant protein/liter of culture) of recombinant proteinsoften results in the expressed protein being found in inclusion bodiesin the host cells. A soluble protein is a protein which is not found inan inclusion body inside the host cell or is found both in the cytoplasmand in inclusion bodies and in this case the protein may be present athigh or low levels in the cytoplasm.

[0098] “Peptide nucleic acid” as used herein, refers to a molecule whichcomprises an oligomer to which an amino acid residue, such as lysine,and an amino group have been added. These small molecules, alsodesignated anti-gene agents, stop transcript elongation by binding totheir complementary strand of nucleic acid (Nielsen, P. E. et. al.,Anticancer Drug Des., 8:53-63 [1993]).

[0099] The term “hybridization” as used herein, refers to any process bywhich a strand of nucleic acid binds with a complementary strand throughbase pairing. Hybridization and the strength of hybridization (i.e., thestrength of the association between the nucleic acids) is impacted bysuch factors as the degree of complementary between the nucleic acids,stringency of the conditions involved, the T_(m) of the formed hybrid,and the G:C ratio within the nucleic acids.

[0100] As used herein, the term “T_(m)” is used in reference to the“melting temperature.” The melting temperature is the temperature atwhich a population of double-stranded nucleic acid molecules becomeshalf dissociated into single strands. The equation for calculating theT_(m) of nucleic acids is well known in the art. As indicated bystandard references, a simple estimate of the T_(m) value may becalculated by the equation; T_(m)=81.5+0.41(% G+C), when a nucleic acidis in aqueous solution at 1 M NaCl (See e.g., Anderson and Young,Quantitative Filter Hybridization, in Nucleic Acid Hybridization[1985]). Other references include more sophisticated computations whichtake structural as well as sequence characteristics into account for thecalculation of T_(m).

[0101] The term “hybridization complex,” as used herein, refers to acomplex formed between two nucleic acid sequences by virtue of theformation of hydrogen binds between complementary G and C bases andbetween complementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., C₀t or R₀tanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized on a solid support (e.g.,membranes, filters, chips, pins or glass slides to which cells have beenfixed for in situ hybridization).

[0102] The terms “complementary” or “complementarity” as used herein,refer to the natural binding of polynucleotides under permissive saltand temperature conditions by base-pairing. For example, for thesequence “A-G-T” binds to the complementary sequence “T-C-A”.Complementarity between two single-stranded molecules may be “partial”,in which only some of the nucleic acids bind, or it may be complete whentotal complementarity exists between the single stranded molecules. Thedegree of complementarity between nucleic acid strands has significanteffects on the efficiency and strength of hybridization between nucleicacid strands. This is of particular importance in amplificationreactions, which depend upon binding between nucleic acids strands.

[0103] The term “homology,” as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology(i.e., identity). A partially complementary sequence is one that atleast partially inhibits an identical sequence from hybridizing to atarget nucleic acid; it is referred to using the functional term“substantially homologous.” The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or Northern blot, solutionhybridization and the like) under conditions of low stringency. Asubstantially homologous sequence or probe will compete for and inhibitthe binding (i.e., the hybridization) of a completely homologoussequence or probe to the target sequence under conditions of lowstringency. This is not to say that conditions of low stringency aresuch that non-specific binding is permitted; low stringency conditionsrequire that the binding of two sequences to one another be a specific(i.e., selective) interaction. The absence of non-specific binding maybe tested by the use of a second target sequence which lacks even apartial degree of complementarity (e.g., less than about 30% identity);in the absence of non-specific binding, the probe will not hybridize tothe second non-complementary target sequence. When used in reference toa single-stranded nucleic acid sequence, the term “substantiallyhomologous” refers to any probe which can hybridize (i.e., it is thecomplement of) the single-stranded nucleic acid sequence underconditions of low stringency as described.

[0104] As known in the art, numerous equivalent conditions may beemployed to comprise either low or high stringency conditions. Factorssuch as the length and nature (DNA, RNA, base composition) of thesequence, nature of the target (DNA, RNA, base composition, presence insolution or immobilization, etc.), and the concentration of the saltsand other components (e.g., the presence or absence of formamide,dextran sulfate and/or polyethylene glycol) are considered and thehybridization solution may be varied to generate conditions of eitherlow or high stringency different from, but equivalent to, the abovelisted conditions.

[0105] As used herein the term “stringency” is used in reference to theconditions of temperature, ionic strength, and the presence of othercompounds such as organic solvents, under which nucleic acidhybridizations are conducted. With “high stringency” conditions, nucleicacid base pairing will occur only between nucleic acid fragments thathave a high frequency of complementary base sequences. Thus, conditionsof “weak” or “low” stringency are often required with nucleic acids thatare derived from organisms that are genetically diverse, as thefrequency of complementary sequences is usually less.

[0106] Low stringency conditions comprise conditions equivalent tobinding or hybridization at 42° C. in a solution consisting of 5×SSPE(43.8 g/l NaCl, 6.9 g/l NaH₂PO₄.H₂O and 1.85 g/l EDTA, pH adjusted to7.4 with NaOH), 0.1% SDS, 5× Denhardt's reagent (50× Denhardt's containsper 500 ml: 5 g Ficoll (Type 400, Pharmacia), 5 g BSA [Fraction V;Sigma]) and 100 μg/ml denatured salmon sperm DNA followed by washing ina solution comprising 5×SSPE, 01% SDS at 42° C. when a probe of about500 nucleotides in length is employed.

[0107] The art knows well that numerous equivalent conditions may beemployed to comprise low stringency conditions; factors such as thelength and nature (DNA, RNA, base composition) of the probe and natureof the target (DNA, RNA, base composition, present in solution orimmobilized, etc.) and the concentration of the salts and othercomponents (e.g., the presence or absence of formamide, dextran sulfate,polyethylene glycol) are considered and the hybridization solution maybe varied to generate conditions of low stringency hybridizationdifferent from, but equivalent to, the above listed conditions. Inaddition, the art knows conditions which promote hybridization underconditions of high stringency (e.g., increasing the temperature of thehybridization and/or wash steps, the use of formamide in thehybridization solution, etc.).

[0108] The term “antisense,” as used herein, refers to nucleotidesequences which are complementary to a specific DNA or RNA sequence. Theterm “antisense strand” is used in reference to a nucleic acid strandthat is complementary to the “sense” strand. Antisense molecules may beproduced by any method, including synthesis by ligating the gene(s) ofinterest in a reverse orientation to a viral promoter which permits thesynthesis of a complementary strand. Once introduced into a cell, thistranscribed strand combines with natural sequences produced by the cellto form duplexes. These duplexes then block either the furthertranscription or translation. In this manner, mutant phenotypes may begenerated. The designation “negative” is sometimes used in reference tothe antisense strand, and “positive” is sometimes used in reference tothe sense strand.

[0109] The term also is used in reference to RNA sequences which arecomplementary to a specific RNA sequence (e.g., mRNA). Included withinthis definition are antisense RNA (“asRNA”) molecules involved in generegulation by bacteria. Antisense RNA may be produced by any method,including synthesis by splicing the gene(s) of interest in a reverseorientation to a viral promoter which permits the synthesis of a codingstrand. Once introduced into an embryo, this transcribed strand combineswith natural mRNA produced by the embryo to form duplexes. Theseduplexes then block either the further transcription of the mRNA or itstranslation. In this manner, mutant phenotypes may be generated. Theterm “antisense strand” is used in reference to a nucleic acid strandthat is complementary to the “sense” strand. The designation. (−) (i.e.,“negative”) is sometimes used in reference to the antisense strand withthe designation (+) sometimes used in reference to the sense (i.e.,“positive”) strand.

[0110] A gene may produce multiple RNA species which are generated bydifferential splicing of the primary RNA transcript. cDNAs that aresplice variants of the same gene will contain regions of sequenceidentity or complete homology (representing the presence of the sameexon or portion of the same exon on both cDNAs) and regions of completenon-identity (for example, representing the presence of exon “A” on cDNA1 wherein cDNA 2 contains exon “B” instead). Because the two cDNAscontain regions of sequence identity they will both hybridize to a probederived from the entire gene or portions of the gene containingsequences found on both cDNAs; the two splice variants are thereforesubstantially homologous to such a probe and to each other.

[0111] “Transformation,” as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. It may occur undernatural or artificial conditions using various methods well known in theart. Transformation may rely on any known method for the insertion offoreign nucleic acid sequences into a prokaryotic or eukaryotic hostcell. The method is selected based on the host cell being transformedand may include, but is not limited to, viral infection,electroporation, lipofection, and particle bombardment. Such“transformed” cells include stably transformed cells in which theinserted DNA is capable of replication either as an autonomouslyreplicating plasmid or as part of the host chromosome. The term“transfection” as used herein refers to the introduction of foreign DNAinto eukaryotic cells. Transfection may be accomplished by a variety ofmeans known to the art including calcium phosphate-DNA co-precipitation,DEAE-dextran-mediated transfection, polybrene-mediated transfection,electroporation, microinjection, liposome fusion, lipofection,protoplast fusion, retroviral infection, and biolistics. Thus, the term“stable transfection” or “stably transfected” refers to the introductionand integration of foreign DNA into the genome of the transfected cell.The term “stable transfectant”

in insect cells. For example, these vectors find use in expressionsystems for recombinant proteins that require eukaryotic processingsystems. It is intended that the present invention encompassbaculovirus-derived vectors, as well as vectors derived from otherviruses capable of infecting invertebrate cells. In preferredembodiments, the vectors are used to infect insect cells.

[0112] As used herein, the term “selectable marker” refers to the use ofa gene which encodes an enzymatic activity that confers the ability togrow in medium lacking what would otherwise be an essential nutrient(e.g., the HIS3 gene in yeast cells); in addition, a selectable markermay confer resistance to an antibiotic or drug upon the cell in whichthe selectable marker is expressed. Selectable markers may be“dominant”; a dominant selectable marker encodes an enzymatic activitywhich can be detected in any eukaryotic cell line. Examples of dominantselectable markers include the bacterial aminoglycoside 3′phosphotransferase gene (also referred to as the neo gene) which confersresistance to the drug G418 in mammalian cells, the bacterial hygromycinG phosphotransferase (hyg) gene which confers resistance to theantibiotic hygromycin and the bacterial xanthine-guanine phosphoribosyltransferase gene (also referred to as the gpt gene) which confers theability to grow in the presence of mycophenolic acid. Other selectablemarkers are not dominant in that there use must be in conjunction with acell line that lacks the relevant enzyme activity. Examples ofnon-dominant selectable markers include the thymidine kinase (tk) genewhich is used in conjunction with tk⁻ cell lines, the CAD gene which isused in conjunction with CAD-deficient cells and the mammalianhypoxanthine-guanine phosphoribosyl transferase (hprt) gene which isused in conjunction with hprt⁻ cell lines. A review of the use ofselectable markers in mammalian cell lines is provided in Sambrook, J.et. al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold SpringHarbor Laboratory Press, New York (1989) pp.16.9-16.15.

[0113] As used herein, the term “vector” is used in reference to nucleicacid molecules that transfer DNA segment(s) from one cell to another.The term “vehicle” is sometimes used interchangeably with “vector.”

[0114] The term “expression vector” as used herein refers to arecombinant DNA molecule containing a desired coding sequence andappropriate nucleic acid sequences necessary for the expression of theoperably linked coding sequence in a particular host organism. Nucleicacid sequences necessary for expression in prokaryotes usually include apromoter, an operator (optional), and a ribosome binding site, oftenalong with other sequences. Eukaryotic cells are known to utilizepromoters, enhancers, and termination and polyadenylation signals.

[0115] The terms “in operable combination,” “in operable order,” and“operably linked” as used herein refer to the linkage of nucleic acidsequences in such a manner that a nucleic acid molecule capable ofdirecting the transcription of a given gene and/or the synthesis of adesired protein molecule is produced. The term also refers to thelinkage of amino acid sequences in such a manner so that a functionalprotein is produced.

[0116] As used herein, the term “amplifiable nucleic acid” is used inreference to nucleic acids which may be amplified by any amplificationmethod. It is contemplated that “amplifiable nucleic acid” will usuallycomprise “sample template.”

[0117] As used herein, the term “sample template” refers to nucleic acidoriginating from a sample which is analyzed for the presence of “target”(defined below). In contrast, “background template” is used in referenceto nucleic acid other than sample template which may or may not bepresent in a sample. Background template is most often inadvertent. Itmay be the result of carryover, or it may be due to the presence ofnucleic acid contaminants sought to be purified away from the sample.For example, nucleic acids from organisms other than those to bedetected may be present as background in a test sample.

[0118] As used herein, the term “primer” refers to an oligonucleotide,whether occurring naturally as in a purified restriction digest orproduced synthetically, which is capable of acting as a point ofinitiation of synthesis when placed under conditions in which synthesisof a primer extension product which is complementary to a nucleic acidstrand is induced, (i.e., in the presence of nucleotides and an inducingagent such as DNA polymerase and at a suitable temperature and pH). Theprimer is preferably single stranded for maximum efficiency inamplification but may alternatively be double stranded. If doublestranded, the primer is first treated to separate its strands beforebeing used to prepare extension products. Preferably, the primer is anoligodeoxyribonucleotide. The primer must be sufficiently long to primethe synthesis of extension products in the presence of the inducingagent. The exact lengths of the primers will depend on many factors,including temperature, source of primer and the use of the method.

[0119] As used herein, the term “probe” refers to an oligonucleotide(i.e., a sequence of nucleotides), whether occurring naturally as in apurified restriction digest or produced synthetically, recombinantly orby PCR amplification, which is capable of hybridizing to anotheroligonucleotide of interest. A probe may be single-stranded ordouble-stranded. Probes are useful in the detection, identification andisolation of particular gene sequences. It is contemplated that anyprobe used in the present invention will be labelled with any “reportermolecule,” so that is detectable in any detection system, including, butnot limited to enzyme (e.g. ELISA, as well as enzyme-based histochemicalassays), fluorescent, radioactive, and luminescent systems. It is notintended that the present invention be limited to any particulardetection system or label.

[0120] As used herein, the term “target” when used in reference to thepolymerase chain reaction, refers to the region of nucleic acid boundedby the primers used for polymerase chain reaction. Thus, the “target” issought to be sorted oat from other nucleic acid sequences. A “segment”is defined as a region of nucleic acid within the target sequence.

[0121] As used herein, the term “polymerase chain reaction” (“PCR”)refers to the method of K. B. Mullis U.S. Pat. Nos. 4,683,195,4,683,202, and 4,965.188, hereby incorporated by reference, whichdescribe a method for increasing the concentration of a segment of atarget sequence in a mixture of genomic DNA without cloning orpurification. This process for amplifying the target sequence consistsof introducing a large excess of two oligonucleotide primers to the DNAmixture containing the desired target sequence, followed by a precisesequence of thermal cycling in the presence of a DNA polymerase. The twoprimers are complementary to their respective strands of the doublestranded target sequence. To effect amplification, the mixture isdenatured and the primers then annealed to their complementary sequenceswithin the target molecule. Following annealing, the primers areextended with a polymerase so as to form a new pair of complementarystrands. The steps of denaturation, primer annealing and polymeraseextension can be repeated many times (i.e., denaturation, annealing andextension constitute one “cycle”; there can be numerous “cycles”) toobtain a high concentration of an amplified segment of the desiredtarget sequence. The length of the amplified segment of the desiredtarget sequence is determined by the relative positions of the primerswith respect to each other, and therefore, this length is a controllableparameter. By virtue of the repeating aspect of the process, the methodis referred to as the “polymerase chain reaction” (hereinafter “PCR”).Because the desired amplified segments of the target sequence become thepredominant sequences (in terms of concentration) in the mixture, theyare said to be “PCR amplified”.

[0122] With PCR, it is possible to amplify a single copy of a specifictarget sequence in genomic DNA to a level detectable by severaldifferent methodologies (eg., hybridization with a labeled probe;incorporation of biotinylated primers followed by avidin-enzymeconjugate detection; incorporation of ³²P-labeled deoxynucleotidetriphosphates, such as dCTP or dATP, into the amplified segment). Inaddition to genomic DNA, any oligonucleotide sequence can be amplifiedwith the appropriate set of primer molecules. In particular theamplified segments created by the PCR process itself are, themselves,efficient templates for subsequent PCR amplifications.

[0123] “Amplification” is a special case of nucleic acid replicationinvolving template specificity. It is to be contrasted with non-specifictemplate replication (i.e., replication that is template-dependent butnot dependent on a specific template). Template specificity is heredistinguished from fidelity of replication (i.e., synthesis of theproper polynucleotide sequence) and nucleotide (ribo- or deoxyribo-)specificity. Template specificity is frequently described in terms of“target” specificity. Target sequences are “targets” in the sense thatthey are sought to be sorted out from other

[0124] As used herein, the terms “restriction endonucleases” and“restriction enzymes” refer to bacterial enzymes, each of which cutdouble-stranded DNA at or near a specific nucleotide sequence.

[0125] As used herein, the term “recombinant DNA molecule” as usedherein refers to a DNA molecule which is comprised of segments of DNAjoined together by means of molecular biological techniques.

[0126] DNA molecules are said to have “5′ ends” and “3′ ends” becausemononucleotides are reacted to make oligonucleotides in a manner suchthat the 5′ phosphate of one mononucleotide pentose ring is attached tothe 3′ oxygen of its neighbor in one direction via a phosphodiesterlinkage. Therefore, an end of an oligonucleotides referred to as the “5′end” if its 5′ phosphate is not linked to the 3′ oxygen of amononucleotide pentose ring and as the “3′ end” if its 3′ oxygen is notlinked to a 5′ phosphate of a subsequent mononucleotide pentose ring. Asused herein, a nucleic acid sequence, even if internal to a largeroligonucleotide, also may be said to have 5′ and 3′ ends. In either alinear or circular DNA molecule, discrete elements are referred to asbeing “upstream” or 5′ of the “downstream” or 3′ elements. Thisterminology reflects the fact that transcription proceeds in a 5′ to 3′fashion along the DNA strand. The promoter and enhancer elements whichdirect transcription of a linked gene are generally located 5′ orupstream of the coding region. However, enhancer elements can exerttheir effect even when located 3′ of the promoter element and the codingregion. Transcription termination and polyadenylation signals arelocated 3′ or downstream of the coding region.

[0127] As used herein, the term “an oligonucleotide having a nucleotidesequence encoding a gene” means a nucleic acid sequence comprising thecoding region of a gene or in other words the nucleic acid sequencewhich encodes a gene product. The coding region may be present in eithera cDNA, genomic DNA or RNA form. When present in a DNA form, theoligonucleotide may be single-stranded (i.e., the sense strand) ordouble-stranded. Suitable control elements such as enhancers/promoters,splice junctions, polyadenylation signals, etc. may be placed in closeproximity to the coding region of the gene if needed to permit properinitiation of transcription and/or

[0128] As used herein, the term “promoter/enhancer” denotes a segment ofDNA which contains sequences capable of providing both promoter andenhancer functions (i.e., the functions provided by a promoter elementand an enhancer element, as discussed above). For example, the longterminal repeats of retroviruses contain both promoter and enhancerfunctions. The enhancer/promoter may be “endogenous,” “exogenous,” or“heterologous.” An “endogenous” enhancer/promoter is one which isnaturally linked with a given gene in the genome. An “exogenous” or“heterologous” enhancer/promoter is one which is placed in juxtapositionto a gene by means of genetic manipulation (i.e., molecular biologicaltechniques), such that transcription of that gene is directed by thelinked enhancer/promoter.

[0129] The presence of “splicing signals” on an expression vector oftenresults in higher levels of expression of the recombinant transcript.Splicing signals mediate the removal of introns from the primary RNAtranscript and consist of a splice donor and acceptor site (See e.g., J.Sambrook et at, Molecular Cloning: A Laboratory Manual, 2nd ed., ColdSpring Harbor Laboratory Press, New York [1989], pp. 16.7-16.8). Acommonly used splice donor and acceptor site is the splice junction fromthe 16S RNA of SV40.

[0130] Efficient expression of recombinant DNA sequences in eukaryoticcells requires expression of signals directing the efficient terminationand polyadenylation of the resulting transcript. Transcriptiontermination signals are generally found downstream of thepolyadenylation signal and are a few hundred nucleotides in length. Theterm “poly A site” or “poly A sequence,” as used herein, denotes a DNAsequence that directs both the termination and polyadenylation of thenascent RNA transcript. Efficient polyadenylation of the recombinanttranscript is desirable, as transcripts lacking a poly A tail areunstable and are rapidly degraded. The poly A signal utilized in anexpression vector may be “heterologous” or “endogenous.” An endogenouspoly A signal is one that is found naturally at the 3′ end of the codingregion of a given gene in the genome. An heterologous poly A signal isone which is isolated from one gene and placed 3′ to another gene. Acommonly used heterologous poly A signal is the SV40 poly A signal. TheSV40 poly A signal is contained on a 237 bp BamHI/BclI restrictionfragment, and directs both termination and polyadenylation (S. Sambrook,supra, at 16.6-16.7).

[0131] Eukaryotic expression vectors may also contain “viral replicons,”or “viral origins of replication.” Viral replicons are viral DNAsequences which allow for the extrachromosomal replication of a vectorin a host cell expressing the appropriate replication factors.

[0132] As used herein, the terms “nucleic acid molecule encoding,” “DNAsequence encoding,” and “DNA encoding” refer to the order or sequence ofdeoxyribonucleotides along a strand of deoxyribonucleic acid. The orderof these deoxyribonucleotides determines the order of amino acids alongthe polypeptide (protein) chain. The DNA sequence thus codes for theamino acid sequence.

[0133] The term “Southern blot” refers to the analysis of DNA on agaroseor acrylamide gels to fractionate the DNA according to size followed bytransfer of the DNA from the gel to a solid support, such asnitrocellulose or a nylon membrane. The immobilized DNA is then probedwith a labeled probe to detect DNA species complementary to the probeused. The DNA may be cleaved with restriction enzymes prior toelectrophoresis. Following electrophoresis, the DNA may be partiallydepurinated and denatured prior to or during transfer to the solidsupport. Southern blots are a standard tool of molecular biologists (Seee.g., J. Sambrook et at, supra at pp 9.31-9-58).

[0134] The term “Northern blot” as used herein refers to the analysis ofRNA by electrophoresis of RNA on agarose gels to fractionate the RNAaccording to size followed by transfer of the RNA from the gel to asolid support, such as nitrocellulose or a nylon membrane. Theimmobilized RNA is then probed with a labeled probe to detect RNAspecies complementary to the probe used. Northern blots are a standardtool of molecular biologists (See e.g., Sambrook et al., supra at pp.7.39-7.52).

[0135] The term “isolated” when used in relation to a nucleic acid, asin “an isolated oligonucleotide” refers to a nucleic acid sequence thatis identified and separated from at least one contaminant nucleic acidwith which it is ordinarily associated in its natural source. Isolatednucleic acid is such present in a form or setting that is different fromthat in which it is found in nature (e.g., in an expression vector). Incontrast, non-isolated nucleic acids are nucleic acids such as DNA andRNA found in the state they exist in nature. For example, a given DNAsequence (e.g., a gene) is found on the host cell chromosome inproximity to neighboring genes; RNA sequences, such as a specific mRNAsequence encoding a specific protein, are found in the cell as a mixturewith numerous other mRNAs which encode a multitude of proteins. However,isolated nucleic acid encoding a mammalian F-box protein includes, byway of example, such nucleic acid in cells ordinarily expressing anF-box protein where the nucleic acid is in a chromosomal locationdifferent from that of natural cells, or is otherwise flanked by adifferent nucleic acid sequence than that found in nature. The isolatednucleic acid or oligonucleotide may be present in single-stranded ordouble-stranded form. When an isolated nucleic acid or oligonucleotideis to be utilized to express a protein, the oligonucleotide will containat a minimum the sense or coding strand (i.e., the oligonucleotide maysingle-stranded), but may contain both the sense and anti-sense strands(i.e., the oligonucleotide may be double-stranded).

[0136] As used herein, the term “immunogen” refers to a substance,compound, molecule, or other moiety which stimulates the production ofan immune response. The term “antigen” refers to a substance, compound,molecule, or other moiety that is capable of reacting with products ofthe immune response. For example, F-box proteins may be used asimmunogens to elicit an immune response in an animal to produceantibodies directed against the subunit used as an immunogen. Thesubunit may then be used as an antigen in an assay to detect thepresence of anti-F-box protein antibodies in the serum of the immunizedanimal. It is not intended that the present invention be limited toantigens or immunogens consisting solely of one protein (i.e., it isintended that the present invention encompass complexes). Nor is itintended that the present invention be limited to any particularantigens or immunogens.

[0137] The term “antigenic determinant,” as used herein, refers to thatportion of a molecule (i.e., an antigen) that makes contact with aparticular antibody (i.e., an

vitro (Cole et al., in Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, pp. 77-96 [1985]).

[0138] According to the invention, techniques described for theproduction of single chain antibodies (U.S. Pat. No. 4,946,778; hereinincorporated by reference) can be adapted to produce F-boxprotein-specific single chain antibodies. An additional embodiment ofthe invention utilizes the techniques described for the construction ofFab expression libraries (Huse et al., Science 246:1275-1281 [1989]) toallow rapid and easy identification of monoclonal Fab fragments with thedesired specificity for F-box proteins

[0139] Antibody fragments which contain the idiotype (antigen bindingregion) of the antibody molecule can be generated by known techniques.For example, such fragments include but are not limited to: the F(ab′)2fragment which can be produced by pepsin digestion of the antibodymolecule; the Fab′ fragments which can be generated by reducing thedisulfide bridges of the F(ab′)2 fragment, and the Fab fragments whichcan be generated by treating the antibody molecule with papain and areducing agent.

[0140] In the production of antibodies, screening for the desiredantibody can be accomplished by techniques known in the art (e.g.,radioimmunoassay, ELISA [enzyme-linked immunosorbant assay], “sandwich”immunoassays, immunoradiometric assays, gel diffusion precipitinreactions, immunodiffusion assays, in situ immunoassays [using colloidalgold, enzyme or radioisotope labels, for example], Western Blots,precipitation reactions, agglutination assays (e.g., gel agglutinationassays, hemagglutination assays, etc.), complement fixation assays,immunofluorescence assays, protein A assays, and immunoelectrophoresisassays, etc.

[0141] As used herein the term “immunogenically-effective amount” refersto that amount of an immunogen required to invoke the production ofprotective levels of antibodies in a host upon vaccination.

[0142] As used herein, the term “reporter reagent” or “reportermolecule” is used in reference to compounds which are capable ofdetecting the presence of antibody bound to antigen. For example, areporter reagent may be a colorimetric substance which is attached to anenzymatic substrate. Upon binding of antibody and antigen, the enzymeacts on its substrate and causes the production of a color. Otherreporter reagents include, but are not limited to fluorogenic andradioactive compounds or molecules.

[0143] As used herein the term “signal” is used in reference to theproduction of a sign that a reaction has occurred, for example, bindingof antibody to antigen. It is contemplated that signals in the form ofradioactivity, fluorogenic reactions, and enzymatic reactions will beused with the present invention. The signal may be assessedquantitatively as well as qualitatively.

[0144] As used herein the term “NF-κB regulatory factors” refers to anyfactors (e.g., proteins, enzymes, peptides, small molecules, and nucleicacids) involved in the regulation of NF-κB signalling pathways. Forexample, such factors include, but are not limited to, F-box proteins,IκBS, IKKs, and agonists, antagonists, and cofactors that interact withthese factors. It is contemplated that the NF-κB regulatory factors caneither directly or indirectly (e.g., through other factors) bind to atarget of interest (e.g., a slimb protein).

GENERAL DESCRIPTION OF THE INVENTION

[0145] The present invention provides compositions and methods for geneidentification, as well as drug discovery and assessment. The presentinvention provides components of an E3 complex involved inubiquitination of cell cycle regulators and other proteins, as well asmembers of a class of proteins that directly function in recognition ofubiquitination targets. These compositions are involved in proteindegradation pathways associated with the eukaryotic cell cycle, amongothers.

[0146] Protein degradation is a commonly employed mechanism for thecontrol of protein abundance. It is also a particularly effective methodfor promoting unidirectional cell cycle transitions because of itsrapidity and irreversibility. Three major transitions (i.e., entry intoS phase, separation of sister chromatids, and exit from mitosis),require the degradation of specific proteins via the ubiquitin-26Sproteosome pathway (reviewed in King et al., Science 274:1652-1659[1996]). Ubiquitin is a relatively small protein (approximately 76 aminoacid residues) found in all cells of higher organisms. Ubiquitin playsmajor roles in intracellular protein degradation and histonemodification.

[0147] Thus, ubiquitination is an important mechanism used to regulateprotein abundance. However, until the development of the presentinvention, the specificity of target selection for ubiquitin dependentproteolysis was largely unknown. Central to this process are the E3swhich confer substrate specificity on the ubiquitination reaction andare therefore likely points for regulation. The present inventionprovides methods for producing ubiquitinated Sic1 in vivo and in vitrousing recombinant proteins. The present invention also providescompositions and methods for the development of drugs and othercompounds effective in correcting abnormalities in protein degradation,based on the demonstration that 1) Cdc53, Skp1, and Cdc4 form afunctional E3 ubiquitin ligase complex that works together with the E2Cdc34 to ubiquitinate Sic1; 2) Cdc4 acts as a receptor forphosphorylated Sic1 recognition; and 3) the sole function Cln/Cdc28kinases in this process is to phosphorylate Sic1, allowing recognitionby Cdc4. Importantly, it was found that distinct F-box proteins canselectively recognize different ubiquitination substrates in aphosphorylation-dependent manner.

[0148] The formation of ubiquitin-protein conjugates in proteindegradation pathways involves three components that participate in acascade of ubiquitin transfer reactions: a ubiquitin activating enzyme(E1), a ubiquitin conjugating enzyme (E2), and a specificity factor (E3)(Hershko et al., J. Biol. Chem., 267:8807-8812 [1983]). Ubiquitin isactivated as a thiol-ester on E1 in an ATP-dependent reaction,transferred to an E2 as a thiol ester and ultimately conjugated to thetarget protein in conjunction with an E3, which functions in substraterecognition and in some instances may serve as a thiol-ubiquitin carrier(Scheffner et al., Cell 75:495-505 [1993]; and Scheffner et al., Nature373:81-83 [1995]). Together, these enzymes polyubiquitinate lysineresidues in target proteins through formation of isopeptide bonds withubiquitin, leading to recognition by the 26S proteosome. Thisassociation eventually results in the degradation of the target protein.

[0149] While E1 and E2 proteins can be identified through sequencesimilarity, this is not yet generally true for E3 proteins. Thus, thepresent invention provides previously unreported methods andcompositions. This is significant as the identity of E3 components are acentral issue in cell cycle control, among other processes, because theyare potential regulators of both the timing of ubiquitination and theselection of substrates. Prior to the development of the presentinvention, much of the prior knowledge of E3s was provided by analysisof the HECT domain protein E6-AP which functions as a ubiquitin-ligasefor p53, (Huibregtse et al., Proc. Natl. Acad. Sci. USA 92:2563-2567[1995]; and Scheffner et al., Nature 373:81-83 [1995]); and the anaphasepromoting complex (APC), which functions in the destruction of mitoticcyclins and proteins involved in sister chromatid cohesion (reviewed inKing et al., [1996] supra). These APC substrates contain a destructionbox motif, although precisely how the timing and selection of substratesby the APC is achieved is unknown. In contrast, timing of ubiquitinationof a variety of non-APC substrates is thought to be regulated in part bythe phosphorylation of the substrate itself. PEST sequences (i.e.,sequences that are rich in proline, glutamic acid, serine and threonine)are frequently found in unstable proteins such as cyclins and containsites of phosphorylation (Rogers et al., Science 234:364-368 [1986]).Phosphorylation of specific residues has been implicated in thedestruction of G1 cyclins in yeast and mammalian cells (Tyers et al.,EMBO J., 11:1773-84 [1992]; Lanker et al., Science 271:1597-1601 [1996];Clurman et al., Genes Dev., 10:1979-1990 [1996]; Diehl et al., GenesDev., 11:957-972 [1997]; and Won and Reed, EMBO J., 15:4182-4193[1996]), and the cyclin-kinase inhibitor (CKI) p27 (Sheaff et al., GenesDev., 11:1464-1478 [1997]).

[0150] In S. cerevisiae, entry into S-phase requires activation of theCdc28 kinase by G1 cyclins (Cln1, Cln2, and Cln3) and S-phase cyclins(Clb5 and Clb6) (Nasmyth, Curr. Opin. Cell Biol., 5:166-179 [1993]).Although both Cln/Cdc28 and Clb/Cdc28 complexes assemble during G1,Clb/Cdc28 is sequestered in an inactive form through association withthe CK1 p40Sic1 (Mendenhall, Science 259:216-219 [1993]; and Schwob et.al., Cell 79: 233-244 [1994]). Sic1 levels vary in the cell cycle,sharply decreasing at the G1/S transition, and this correlates withactivation of Clb5/Cdc28. The decrease in Sic1 levels depends on the E2Cdc34, suggesting that ubiquitination triggers Sic1 destruction (Schwobet al., Cell 79:233-244 [1994]). Sic1 destruction also requires CLN andCDC28 function; elimination of Sic1 defines the threshold requirementfor Cln/Cdc28 activity in S-phase entry (Schneider et al., Science 272:560-562 [1996]; Schwob et al., [1994], supra; and Tyers, Proc. Natl.Acad. Sci. U.S.A. 93:7772-7776 [1996]). Although Sic1 is aphosphoprotein (Schneider et al., Science 272:560-562 [1996]), it is notknown whether Cln/Cdc28 complexes directly phosphorylate Sic1 or whetherphosphorylation plays another, perhaps indirect, role in Sic1destruction. The development of the present invention provides methodsand compositions to resolve these questions.

[0151] Three other genes, SKP1, CDC53, and CDC4, are also required forS-phase entry (Schwob et al., 1994, supra; and Bai et al., Cell86:263-274 [1996]). These genes, together with CDC34, show a pattern ofsuppression and enhancement consistent with roles in a common process;conditional alleles of these genes cause arrest with unreplicated DNAand multiple buds (Yochem and Byers, J. Mol. Biol., 195:233-245 [1987];Goebl et al., J. Mol. Biol., 195:233-245 [1988]; Bai et al., [1996]supra; and Mathias et al., Mol. Cell. Biol., 16:6634-6643 (1996]). Sic1accumulates in cdc34-1, cdc4-1, or skp 1-11 mutants, and deletion ofSIC1 allows such mutants to undergo DNA synthesis (Schwob et al.,[1996], supra; and Bai et al., [1996], supra). Components of the Cdc34pathway have also been implicated in the destruction of a number ofother important regulatory proteins, including Cln2 (Deshaies et al.,EMBO J., 14:303-312 [1995]; Bai et al., [1996] supra; and Willems etal., Cell 86:453-463 [1996]), Cdc6 (Piatti et al., Genes Dev.,10:1516-1531 [1996]), the CKIs Rum1 and Far1 (McKinney et al., GenesDev., 7:833-843 [1993]; and Kominami and Toda, Genes Dev., 11:1548-1560[1997]), and the transcription factor Gcn4 (Kornitzer et al., EMBO J.,33:6021-6030 [1994]). Thus, it is contemplated that Cdc34, Cdc53, Skp1,and Cdc4 are utilized for the destruction of diverse regulatoryproteins. A requirement for Cdc34 for Cln2 ubiquitination has beendemonstrated in crude yeast lysates (Deshaies et al., [1995], supra),but this requirement has been suggested to be indirect (Blondel andMann, Nature 384:279-282 [1996]). Interestingly, SKP1 is also requiredfor the G2/M transition (Bai et al., [1996], supra; and Connelly andHeiter, Cell 86:275-285 [1996]), and has been found to be a component ofthe kinetochore complex CBF3 (Connelly and Fleiter, Cell 86:275-285[1996], supra; and Stemmann and Lechner, EMBO J., 15:3611-3620 [1996]).

[0152] Skp1 binds to Cdc4, and this interaction involves a motif in Cdc4referred to as the F-box (Bai et al., [1996], supra). The F-box motif isfound in a large number of proteins including cyclin F (Bai et al., EMBOJ., 15:3611-3620 [1994]) and the cyclin A/Cdk2-associated protein Skp2(Zhang et al., Cell 82:915-925 [1995]), both of which bind Skp1. The twolargest classes of F-box proteins either contain WD-40 repeats (e.g.,Cdc4) or leucine-rich repeats (LRR) (e.g., Skp2 and Grr1) (Bai et al.,[1996], supra). GRR1 was initially identified as a gene required forglucose repression (Flick and Johnston, Mol. Cell. Biol., 11:5101-12[1991]) but was later also found to be involved in Cln destruction(Barral et al., Genes Dev., 9:399-409 [1995]). The discovery that Skp1is required for the destruction of both Sic1 and Clin2, while Cdc4 andGrr1 were only implicated in the destruction of one of these, led todevelopment of one embodiment of the present invention (i.e., one model)in which F-box proteins function to recognize targets forubiquitination, and Skp1 links these F-box/target complexes to theubiquitination machinery.

[0153] The present invention was developed in a stepwise fashion, withan important aspect being the elucidation of the role of Skp1 and F-boxproteins in ubiquitination through in vitro reconstruction of the Sic1ubiquitination pathway. Sic1 ubiquitination was found to depend uponeach of the proteins implicated in Sic1 destruction in vivo. Forexample, Skp1 recruits Cdc4 into a Cdc53/Cdc34 complex, and enhancesrecognition of Sic1 by Cdc4, with the latter interaction requiring Sic1phosphorylation. In contrast, Grr1 does not interact with Sic1, but doesrecruit phosphorylated Cln1 and Cln2 into Skp1/Cdc53 complexes. Thus,the present invention provides F-box proteins that function asreceptors, which recruit substrates into a Skp1/Cdc53/Cdc34 complex forubiquitination by Cdc34.

[0154] Thus, the present invention provides the function of a class ofproteins referred to as F-box proteins in targeted ubiquitination. Thepresent invention finds utility in methods for developing compounds thataffect ubiquitination. The present invention also provides numerousnovel F-box containing mammalian genes whose encoded proteins arecontemplated to function in processes including, but not limited, totargeted ubiquitination of cellular proteins. Specifically, F-boxproteins function as receptors for proteins to be ubiquitinated.

[0155] As described in the Examples, through a series of experimentsusing a set of defined proteins found in S. cerevisiae, it wasdemonstrated that three proteins (i.e., Cdc53, Skp1, and the F-boxprotein Cdc4) form a complex referred to as an “E3” which functionstogether with an E1 ubiquitin activating enzyme, and the E2 ubiquitinconjugating enzyme Cdc34, to ubiquitinate the Cdk inhibitor Sic1.Recognition of Sic1 by this E3 complex requires that Sic1 bespecifically phosphorylated and phosphorylation may be a generalmechanism used to regulate the timing of ubiquitination of targetproteins. Thus, it is contemplated that compounds that alter thisphosphorylation will in turn, alter the timing of ubiquitination oftarget proteins. Such compounds are contemplated as possible drugs thatdisrupt at least one pathway in which F-box proteins function, and arerequired for biological and/or biochemical processes.

[0156] Cdc53 was found to function as an adapter and link Skp1 to theE2, while Skp1 was found also to function as an adapter and links Cdc53to the F-box protein Cdc4. Cdc4 was found to function as an adapter tolink ubiquitination targets (e.g., Sic1) to the Skp1/Cdc53/Cdc34complex. E1 is not a stable component of the ubiquitination complex, butis required for ubiquitination of the target protein. The F-box proteincontains minimally two protein-protein interaction domains. The F-box isa sequence of 35-45 amino acids and allows the F-box proteins to enterinto complexes with Skp1. F-box proteins also contain additionaldomains, typically, but not necessarily C-terminal to the F-boxsequence, which based on the results with Cdc4 function as recognitioncomponents for ubiquitination substrates. Cdc4 contains C-terminal WD-40repeats. Another F-box protein (Grr1) contains leucine rich repeatswhich are protein-protein interaction domains. Because Skp1simultaneously forms complexes with Cdc53 proteins and an F-box protein,these interactions give rise to formation of an E3 complex. Anyparticular F-box protein may interact simultaneously with both Skp1 andat least one ubiquitination target. F-box proteins may have a singleubiquitination target but it is contemplated that they (i.e., at leastsome F-box proteins) also have multiple in vivo ubiquitination targets.For example, the data obtained for Cdc4 indicate that it is involved inthe destruction of at least two proteins, Sic1 and Cdc6. Thus, thepresent invention provides the necessary components and methods to alterubiquitination of target proteins through the use of new drugs or othercompounds.

[0157] Based on the sequence of the yeast genome, it was determined thatS. cerevisiae contains nine F-box proteins. CDC4 is required for thedestruction of Sic1 and Cdc6, while Grr1 is required for the destructionof the G1 (Cln) cyclins, and MET30 is required for proper control ofmethionine biosynthetic pathways and is predicted to control theabundance of Met4.

[0158] The present invention also provides methods and compositionsuseful to determine the complexity and diversity of mammalian F-boxproteins, as well as the identity of F-box proteins from variousspecies, the protein-protein interaction domains involved, theproteolytic pathways, and regulatory pathways. For example, themammalian proteins (cyclin F, Skp2) contain an F-box and associate withSkp1, but their functions and ubiquitination targets have not beendemonstrated. Cyclin F contains a cyclin box motif C-terminal to theF-box. Skp2 contains a leucine rich motif C-terminal to its F-box. MouseMD6, an additional mammalian F-box containing protein; X54352) is inGenbank but its function is unknown. The present invention provideshuman MD6, with the following sequences: (SEQ ID NO:57)LPLELSFYLLKWLDPQTLLTCCLVSKQWNKVISACTEVW; and (SEQ ID NO:58; AA252600)CTTCCCCTGGAGCTCAGTTTTTATTTGTTAAAATGGCTCGATCCTCAGACTTTACTCACATGCTGCCTCGTCTCTAAACAGTGGAATAAGGTGATAAGTG CCTGTACAGAGGTGTGG.

[0159] Furthermore, the closest homolog of MD6 is MET30; it iscontemplated that MD6 plays a homologous role in methionine biosynthesisin eukaryotes. The present invention provides methods and compositionsto identify the functions and ubiquitination targets of these and otherF-box containing proteins.

[0160] The present invention also provides amino acid and DNA sequenceinformation for eighteen novel F-box-containing human or mouse genes. Aswith Cdc4, Grr1, Skp2, and cyclin F, these novel F-box proteins have thecapacity to associate with Skp1 and to simultaneously interact withother proteins through other protein-protein interaction motifs encodedby regions of their genes other than the F-box. Thus, the presentinvention provides compositions and methods for determining theinteraction of these proteins with other proteins.

[0161] Mammalian Skp1, by analogy with budding yeast, functions as anadapter linking Skp1 to an E2. It is contemplated that cellular proteinsbrought into complexes containing Cdc53 and Skp1 by any one of thesenovel F-box proteins has the potential to be ubiquitinated by an E2(e.g., Cdc34) in combination with an E1. It is further contemplated thatinteraction with an F-box protein may also produce an alternativeregulatory function (e.g., altering subcellular localization of theassociated protein). Thus, the function of F-box proteins is notnecessarily limited to ubiquitination, and the present inventionprovides the methods and compositions to make this determination. It iscontemplated that additional F-box containing genes will be discoveredthrough the use of two-hybrid screens with Skp1 or ubiquitinationtargets as the two-hybrid “bait” (e.g., as described in the Example 6).It is also contemplated that additional F-box genes will be discoveredthrough sequencing of the mammalian genome and sequence analysis, todetermine the homology with existing F-box proteins, such as thoseidentified in the present invention.

[0162] For example, it is contemplated that cell cycle regulators suchas cyclins and cyclin-kinase inhibitors, transcription regulators,proteins involved in DNA replication, and other cellular regulatoryproteins will be identified and/or investigated using the methods andcompositions provided by the present invention. It is furthercontemplated that the present invention will find use in elucidatinginflammatory response and infectious disease processes involving proteindegradation, as well as development of compounds that control (i.e.,either enhance or retard) protein degradation as appropriate, toameliorate the effects of the inflammatory response or disease process.

[0163] Thus, it is also contemplated that F-box proteins are involved inregulatory pathways important for cellular homostasis and/or growthcontrol. In this context, F-box proteins may be involved in theelimination or modification of proteins which positively or negativelyregulate the cell cycle, which positively or negatively regulatetranscription, or which positively or negatively regulate the abundanceof a protein involved in a signaling pathway. Elimination of proteinscould be mediated by the 26S proteosome after targeted ubiquitination bya E3 complex containing an F-box protein. Ubiquitination withoutproteolytic destruction may alter the activity of the target proteineither positively or negatively. Thus, it is contemplated that moleculesthat alter the activities or target specificities of F-box proteins, orthe ability of F-box proteins to enter into macromolecular complexessuch as E3 complexes composed of and F-box protein, a Cdc53 homolog andSkp1, will find utility as pharmaceutical agents for a variety ofdiseases. The present invention provides the compositions and methodsfor the identification of molecules (including but not limited toproteins, peptides, naturally occuring alkaloids, and syntheticalkaloids) which alter the activities, levels, or targets of F-boxproteins.

[0164] For example, disruption of the F-box protein/Skp1 complex isachieved using synthetic molecules, proteins, or peptides which mimicthe F-box sequence or its three dimensional structure and blockassociation of any F-box protein with Skp1. It is contemplated thatblockage of this interaction renders the F-box protein non-functionalwith respect to ubiquitination of its target proteins. Similarly,disruption of such complexes is also achieved with synthetic molecules,proteins, or peptides which specifically bind the F-box of a particularF-box protein. This approach provides specificity for a particularpathway involving a specific F-box. These classes of molecules can beidentified using various methods, including, but not limited to, peptidephage display libraries to identify peptide sequences that bind eitheran F-box sequence of a specific domain in Skp1 involved in interactionwith the F-box. In this method, F-box sequences or Skp1 sequences areimmobilized on solid supports such as a magnetic bead through the use ofbiotinylated F-box or Skp1 sequences and streptavidin coated magneticbeads. Phage display libraries are then bound to the coated magneticbeads and phage binding the beads are isolated and analyzed for bindingsequences.

[0165] A similar method involves the use of two-hybrid screens toidentify proteins or fragments of proteins that bind Skp1 or the F-boxsequence. Such molecules find use in blocking assembly of Skp1/F-boxprotein complexes in vivo and are useful (either directly or asprecursors) in the generation of pharmacological agents.

[0166] In another embodiment, disruption of F-box/target interactionsare also contemplated. In addition to the F-box, F-box containingproteins may also contain an additional interaction domain including butnot limited to WD-40 or leucine rich repeats. For example, F1 Alpha andF2 Beta contain leucine rich repeats. Embodiments of the presentinvention provide methods to identify targets of F-box proteins whichinclude, but are not limited to cyclins, cell cycle regulators,cyclin-kinase inhibitors, β-catenin, IκB, and transcriptionalregulators. It is contemplated that molecules which either block,enhance, or otherwise facilitate association of any target with anyF-box protein are useful as pharmaceutical agents in the treatment ofhuman diseases. The approaches described herein provide examples ofapproaches that would yield peptides, proteins, and naturally occuringor synthetic molecules which can bind target recognition motifs in F-boxproteins or motifs in the target protein responsible for recognizing theF-box protein. It is also contemplated that molecules which bind thesedomains block complex formation and thereby block, accelerate, or alterthe normal function of the F-box protein, which may include (dependingupon the particular F-box protein), but is not limited toubiquitination.

[0167] The present invention also provides experimental strategies todetermine whether molecules identified in these ways can block complexassembly. It is contemplated that binding assays based on immobilizedSkp1 and soluble F-box protein (or vice versa), or immobilized F-boxprotein and soluble target (or vice versa), will be developed in amanner similar to the development of embodiments of the presentinvention (i.e., with Skp1, Cdc4 [an F-box protein] and Sic1 [the targetof Cdc4]). Molecules to be tested for their ability to alter eitherSkp1/F-box protein interaction or F-box protein/target interaction maybe added to binding reactions and the effects of the added agentexamined by determining the fraction of soluble protein bound relativeto that bound in the absence of the agent. It is also contemplated thatsuch an assay be adapted to high throughput screening strategies throughthe use of radiolabeled or otherwise tagged soluble binding protein.

[0168] The present invention also provides evidence for phosphorylationspecific recognition of target proteins and methods for determiningwhether recognition of the target requires that the target bephosphorylated. It is contemplated that agents that block or enhancespecific phosphorylation of target proteins to allow recognition byF-box proteins will be identified through approaches disclosed herein.It is contemplated that such agents will find use as pharmaceuticalagents that increase or decrease the rate of ubiquitination of targetproteins.

[0169] In addition, the present invention finds use in theidentification and development of compounds effective against viralinfection and disease. For example, two viral proteins (adenovirusE3-12.9K and baculovirus ORF11), appear to essentially encode only anF-box, and a SKP1-related gene is present in Chorella virus. As virusessubvert the cell cycle in order to replicate, it is contemplated thatdisruption of the ubiquitin-mediated proteolysis pathway would alsodisrupt viral replication. It is possible that F-box containing virusescan inhibit degradation of specific protein subsets (e.g., cyclins) toenhance their replication, or promote the degradation of specificinhibitory proteins. It is also possible that these proteins may targetthe destruction of proteins that inhibit or kill the virus. The presentinvention finds use in development of compositions and methods toinhibit viral replication by interfering with the ubiquitin-mediatedproteolysis pathway utilized by the virus, as well as by upregulatingthe cellular machinery to enhance proteolysis of viral components. Inparticular, the present invention finds use in identification anddevelopment of compounds effective against immunodeficiency viruses(e.g., human immunodeficiency virus, as well as other viruses such asfeline immunodeficiency virus, bovine immunodeficiency virus, and simianimmunodeficiency virus).

[0170] It is further contemplated that targets of novel human F-boxproteins will be determined by those experienced in the art byapproaches including, but not limited to two-hybrid library screens,immunoprecipitation analysis followed by immunoblotting with antibodiesagainst candidate targets, peptide mapping, mass spectral analysis,peptide sequencing, and/or by screening lambda based expressionlibraries with F-box protein probes.

[0171] For example, the present invention provides an example whereby anovel E3 ubiquitin ligase complex has been identified using the methodsand compositions described herein. In particular, the F-box proteinslimb (TRCP), was found to associate with IκB, providing the potentialto screen for factors that regulate the NF-κB pathway. This hasimportant implications in the regulation and control of cancer and theimmune system, among other important physiological effects.

[0172] The present invention also finds use in investigating thefunction and methods of altering protein targets whose abundance isaltered in disease. For example, cyclins are frequently overexpressed incancer cells. Thus, mutations in F-box proteins involved in cyclindestruction will lead to cyclin accumulation; such cyclin accumulationmay promote inappropriate cell division characteristic of cancer. Thepresent invention also finds utility in the identification of mutationsin F-box genes through various methods, including, but not limitedsequence analysis, Southern blot analysis of DNA, etc. Furthermore, thepresent invention also finds use in assessing alterations in cellularprotein abundance due to overexpression of particular F-box proteins. Itis contemplated that such alterations are associated with particulardiseases. The present invention also finds use in determination ofoverexpression caused by gene amplification in DNA samples from diseasedtissue or individuals through such methods as Southern analysis using aparticular F-box gene as probe.

[0173] Furthermore, the present invention thereby provides methods andcompositions for the detection and analysis of abnormalities inproteolytic functions, as well as methods and compositions for thedevelopment of compounds suitable for use in ameliorating suchabnormalities.

DETAILED DESCRIPTION OF THE INVENTION

[0174] As discussed above, the present invention provides compositionsand methods for gene identification and characterization, as well asdrug discovery and assessment. In particular, the present inventionprovides components of an E3 complex involved in ubiquitination of cellcycle regulators and other proteins, as well as members of a class ofproteins that directly function in recognition of ubiquitination targets(i.e., F-box proteins). These compositions are involved in proteindegradation pathways associated with the eukaryotic cell cycle.

[0175] Assembly of a Complex Containing Cdc53/Skp1/Cdc4 and the E2 Cdc34

[0176] Strong genetic evidence implicated Cdc34, Cdc53, Skp1, and Cdc4as molecules involved in the control of S-phase entry throughdestruction of Sic1. In preliminary work, SKP1 and CDC4 were found toshow reciprocal overproduction suppression of their respectivetemperature sensitive mutants and that Cdc4 physically associated withSkp1. A further search for suppressors using a GAL-driven cDNA libraryrevealed that CDC53 overexpression suppresses skp1-11. Theseobservations, coupled with genetic and physical evidence of aCdc53/Cdc34 interaction resulted in the development of embodiments ofthe present invention.

[0177] The first step in assembling the complexes of interest involvedco-infection of insect cells with various baculovirus expressionvectors. Insect cells were co-infected with various combinations ofbaculoviruses expressing Myc-tagged Cdc53 (Cdc53^(M)), Cdc34, Cdc4, andSkp1. Anti-Myc immune complexes from lysates of these infected cellswere immunoblotted to identify associated proteins (See, FIG. 1A). Asshown in FIG. 1A, in the presence of all four proteins, anti-Cdc53^(M)complexes contained Cdc4, Cdc34, and Skp1 (FIG. 1A, lane 5). However, inthe absence of Skp1, only low levels of Cdc4 were found to bind withCdc53^(M), regardless of the presence of Cdc34 (See, FIG. 1A, lanes 7and 8). To confirm this result, the association of Cdc53^(M) withanti-Cdc4^(F) immune complexes was analyzed. These results indicated theassociation of Cdc53^(M) with anti-Cdc4^(F) immune complexes was alsogreatly enhanced in the presence of Skp1 (See, FIG. 1B). Thus, onefunction of Skp1 is to facilitate association of Cdc53 with Cdc4. Incontrast to Cdc4, both Skp1 and Cdc34 were shown to associate withCdc53^(M) in the absence of other yeast proteins (See, FIG. 1A).Furthermore, it appeared that Cdc53 can simultaneously associate withboth Cdc34 and Skp1, as the association of Gst-Skp1 with Cdc34 isenhanced in the presence of Cdc53^(M) (See, FIG. 1C). These dataindicated that Cdc34, Cdc53, Skp1, and Cdc4 form a multiprotein complex.

[0178] Phosphorylation of Sic1 by Cln/Cdc28 is Required for itsRecognition by a Cdc4/Skp1/Cdc53 Complex

[0179] While previous studies implicated involvement ofCln/Cdc28-dependent phosphorylation in Sic1 degradation (Schwob et al.,Cell 79:233-244 [1994]; Schneider et al., Science 272:560-562 [1996];and Tyers, Proc. Natl. Acad. Sci. U.S.A. 93:7772-7776 [1996]), until thedevelopment of the present invention, it was not clear whether Sic1 wasdirectly phosphorylated by Cln/Cdc28, or whether this phosphorylationwas correlative or causative for subsequent Sic1 degradation (and ifcausative, whether this modification played a role in Sic1 recognitionby the ubiquitination machinery). Nor was it known whether Cln/Cdc28might also directly regulate the activity of the ubiquitinationmachinery. Once the methods to generate and purify Cln1/Gst-Cdc28 andSic1/Clb5/Gst-Cdc28 complexes from insect cells were established invitro during the development of the present invention, the determinationwas made as to whether any of these components might function in Sic1recognition, and if Sic1 phosphorylation plays a role in this process.This aspect of the present invention finds use in providing methods forthe development of drugs or other compounds suitable for preventionand/or treatment of cancers (i.e., uncontrolled cellular growth), aswell as treatment of other diseases associated with abnormalities incell cycle control.

[0180] In order to accomplish this, Sic1 was purified to nearhomogeneity from insect cells by virtue of its association withClb5/Gst-Cdc28 complexes (See, FIG. 2B). Initially, it was believed thatsuch a complex would represent the primary form of Sic1 ubiquitinated invivo. However, it was found that uninhibited Clb5/Cdc28 in thesepreparations phosphorylated Sic1, making it impossible to directlyassess the role of specific phosphorylation by Clns. Therefore, akinase-impaired Gst-Cdc28(K−) containing a mutation in a criticalcatalytic residue (D145N) was used to assemble Sic1 complexes. In suchcomplexes, Sic1 remains essentially unphosphorylated, however the Sic1is readily phosphorylated by Cln1/Cdc28 (See, FIG. 2A). In vitrophosphorylation of Sic resulted in a reduction in its electrophoreticmobility (See, FIG. 2B), reminiscent of that observed with Sic1 in vivo.

[0181] In the absence of Cln1 kinase, the extent of Sic1 phosphorylationwas found to be less than 2% of that of phosphorylated Sic1, but thismodification did not result in alterations in electrophoretic mobility.For simplicity, this weakly phosphorylated form of Sic1 is hereinreferred to as “unphosphorylated Sic1.”

[0182] Cdc4 is the Specificity Factor for Recognition of PhosphorylatedSic1

[0183] Phosphorylated and unphosphorylated Sic1 were used in bindingreactions with anti-Cdc53^(M) immune complexes assembled and purifiedfrom insect cells (See, FIG. 2C). Phosphorylated Sic1 was found toefficiently associate with Cdc53/Skp1/Cdc4 complexes; this associationwas dependent upon the presence of Skp1 (See, FIG. 2C, lanes 6 and 8).Typically 10-20% of the input phosphorylated Sic1 was bound at about 20nM Sic1. In contrast, the extent of binding of unphosphorylated Sic1(See, FIG. 2C, lane 7) was comparable to that observed in control immunecomplexes generated from uninfected cells (See, FIG. 2C, lane 3), andwas less than 1% of the input Sic1. It was also observed that,consistent with the results in FIG. 1, the level of Cdc4 found in immunecomplexes lacking Skp1 were more than 10-fold lower than that found inthe presence of Skp1. Thus, Cdc4 and/or Skp1 function as binding factorsfor Sic1, and association of Sic1 with this complex requiresphosphorylation by Cln1/Cdc28.

[0184] In addition, to directly examine the roles of Skp1 and Cdc4 inSic1 recognition, binding experiments were performed using series ofcomplexes assembled in vivo that contained constant high levels ofFlag-tagged Skp1 (Skp1^(F)), and increasing quantities of Cdc4. Theseexperiments, as described in the Examples, showed that association ofphosphorylated Sic1 with anti-Skp1^(F) immune complexes was absolutelydependent upon the presence of Cdc4 (e.g., compare lanes 3 and 9 of FIG.2E). Moreover, deleting the last three WD-40 repeats from the C-terminusof Cdc4 (Cdc4ΔAWD) abolished its ability to associate withphosphorylated Sic1 (See, FIG. 2E, lanes 10-16). Therefore, theseexperiments indicated that Cdc4 functions as the specificity factor forbinding of phosphorylated Sic1, and the Cdc4-Sic1 interaction requiresan intact WD-40 repeat domain in Cdc4. While Skp1 alone does notinteract with Sic1, it stimulates association of Sic1 with withFlag-Cdc4 (Cdc4^(F)) by about 5-fold (See, FIG. 2D). The weakassociation of Sic1 with Cdc4 alone (See, FIG. 2D, lane 3) may reflectthe participation of an insect cell Skp1 homolog. Although it is notclear if Skp1 physically contacts Sic1 or stabilizes a form of Cdc4compatible with Sic1 binding, and such an understanding is not necessaryin order to use the present invention, these results clearlydemonstrated that there is a positive contribution of Skp1 in theCdc4/Sic1 interaction.

[0185] Sic1 is Ubiquitinated In Vivo

[0186] While the finding that Cdc4, Skp1, and Cdc53 form a complex thatbinds both phosphorylated Sic1 and the E2 Cdc34 was consistent with arole for ubiquitination in the regulation of Sic1 abundance, prior tothe development of the present invention, Sic1 had not been demonstratedto be ubiquitinated in vivo. In order to directly accomplish this,insect cell lysates were generated from either wild type cells or sic1deletion mutants expressing His₆-Ub^(RA) or Ub^(RA) as a negativecontrol, and ubiquitinated proteins purified using Ni⁺² beads (Willemset al., Cell 86:453-463 [1996]) prior to immunoblotting with anti-Sic1antibodies (See, FIG. 3A).

[0187] The K48R mutation in Ub^(RA) blocks polyubiquitination andtherefore recognition by the proteolytic machinery (i.e., proteosomerecognition) (Chau et al., Science 243:1576-1583 [1989]), while the G76Amutation reduces the rate at which hydrolases remove ubiquitinconjugates (Hodgins et al., J. Biol. Chem., 267:8807-8812 [1992]). Aladder of bands recognizable by anti-Sic1 antibodies was detected in theNi⁺²-bead bound proteins from wild type lysates expressing His₆-Ub^(RA)(See, FIG. 3A, lane 8) but not in conjugates derived fromUb^(RA)-expressing cells or a sic1 deletion strain (See, FIG. 3A, lanes5 and 6). This result demonstrates that Sic1 is ubiquitinated in vivo.Thus, the present invention also provides an important therapeutictarget for development of drugs and other compounds for diseaseprevention and/or treatment.

[0188] Reconstitution of the Sic1 Ubiquitination Pathway Using PurifiedProteins

[0189] Once a strategy to generate Cdc4/Skp1/Cdc53 complexes thatrecognized phosphorylated Sic1 was developed, the next step was todetermine whether these complexes can catalyze ubiquitination of Sic1 invitro when supplemented with Cdc34, E1, ATP, and ubiquitin. It wasobserved that in the presence of all reaction components, phosphorylatedSic1 in complexes with Clb5/Cdc28 was efficiently convened to highermolecular weight conjugates detectable with anti-Sic1 antibodies (See,FIG. 5B, lane 6; and FIG. 5C, lane 5). In contrast, unphosphorylatedSic1 was not detectably ubiquitinated. Sic1 ubiquitination absolutelyrequired Cdc34, Cdc4, Cdc53, Skp1, E1 and ubiquitin (See e.g, FIG. 5Band FIG. 5C), as well as yeast Skp1. The pattern of high molecularweight Sic1 conjugates obtained in reactions with ubiquitin wasdifferent from that observed when Gst-Ub^(RA) was used as the ubiquitinsource, (See, FIG. 5C, compare lanes 5 and 11) confirming that the highmolecular weight forms observed were products of ubiquitination. WithGst-Ub^(RA), the Sic1 reaction products were integrated into a ladder ofbands differing by approximately 35 kDa, the size of Gst-Ub^(RA) (See,FIG. 3C, lane 11). Since Gst-Ub^(RA) had a reduced ability to formpolyubiquitin chains, the number of bands observed is likely to reflectthe number of individual lysines ubiquitinated on a single Sic1molecule. The ubiquitination reaction was time-dependent and thereaction efficiency ranged from 10-40% of the input Sic1 protein (Seee.g., FIGS. 3B and 3C). When the reaction was performed with pre-boundSic1, the efficiency was greater than 50%. In addition, it was foundthat greater than 50% of the Sic1 ubiquitin conjugates formed after 60minutes had dissociated from the Cdc4/Skp1/Cdc53 complex. NeitherGst-Cdc28, Clb5, Cdc53, Skp1, or Cdc4 formed ubiquitin conjugates underthe reaction conditions employed, although Cdc34 was ubiquitinated aspreviously reported.

[0190] To test whether Sic1 ubiquitination requires association withClb5/Cdc28 complexes, ubiquitination reactions using Sic1 produced inbacteria were performed both with and without phosphorylation withCln2/Cdc28 (See, FIG. 3D). As in the case of Sic1 assembled in insectcells with Clb5/Cdc28, phosphorylated Sic1 from bacteria was efficientlyubiquitinated with greater than 90% of the Sic1 forming ubiquitinconjugates (See, FIG. 3D, lane 8), and ubiquitination absolutelyrequired Sic1 phosphorylation (i.e., unphosphorylated Sic1 was notubiquitinated; See e.g., FIG. 3D, lane 4). Thus, phosphorylation of Sic1was shown to be required for its recognition by Cdc4 and Skp1.

[0191] Next, it was determined whether Cln/Cdc28, present in smallamounts in the ubiquitination reaction, is also required for additionalsteps in the ubiquitination process (e.g, to phosphorylate theubiquitination machinery). This was accomplished by treating bacterialSic1 with Cln2/Gst-Cdc28 complexes immobilized on GSH-Sepharose beads,removing the complexes from the beads prior to use in ubiquitinationreactions, and determining whether the complexes were free of solublekinase by immunoblotting with anti-HA antibodies (See, FIG. 3D, lane 3).These results indicated that Sic1 phosphorylated in this manner was alsoefficiently ubiquitinated (See, FIG. 3D, lane 9). Thus, these dataindicated that Sic1 phosphorylation constitutes the primary requirementof Cln/Cdc28 kinases in Sic1 ubiquitination in the in vitro reaction.

[0192] Although Sic1 was found to be an inhibitor of Cdc28/Clb5complexes, when the kinase complex contained an excess of Sic1, it wasincapable of phosphorylating Sic1 and converting it into a substrate forubiquitination (FIG. 3E shows the reduced electrophoretic mobility) and³²P incorporation. This Clb5/Cdc28-phosphorylated Sic1 was also asubstrate for ubiquitination (See, FIG. 3E). Although it is notnecessary to understand the mechanisms involved in order to use thepresent invention, overexpression of CLB5 can drive S-phase entry incln- cells and suggests that active Clb5/Cdc28 formed during Sic1destruction may collaborate with Cln/Cdc28 to complete the Sic1ubiquitination process.

[0193] F-box Proteins are Receptors for Ubiquitination Substrates

[0194] The determination that Cdc4 functions in the recognition andubiquitination of phosphorylated Sic1 is consistent with a function ofF-box proteins being recognition of ubiquitination targets. During thedevelopment of the present invention, investigations into whetherspecific F-box proteins could have broad specificity and interact withmultiple targets, or could be relatively restricted in their targetspecificity, perhaps associating with only a single target, wereconducted.

[0195] To elucidate the selectivity of F-box proteins, experiments wereconducted to determine whether substitution of Cdc4 by another F-boxprotein (Grr1) could support Sic1 binding and ubiquitination. Grr1 hasan F-box near its N-terminus and can interact simultaneously with Skp1and Cdc53 when co-expressed in insect cells. Gene 10-tagged Grr1(Grr1¹⁰) was also found to interact simultaneously with Skp1 and Cdc53,when co-expressed in insect cells (See, FIG. 4A). It was found that Grr1and Cdc4 interact with Skp1/Cdc53 in a mutually exclusive manner. Incontrast with Cdc4, however, the Grr1/Cdc53 interaction in insect cellswas not enhanced by co-expression of Skp1, although Skp1 assembled withthese complexes.

[0196] Importantly, Grr1 assembled with Cdc53/Skp1 (i.e.,Cdc53/Skp1/Grr1 complex) was unable to associate with phosphorylatedSic1 and did not support ubiquitination of phosphorylated Sic1 complexesin the in vitro system with purified proteins under conditions whereCdc4 readily facilitates Sic1 binding and ubiquitination (See, FIGS. 4Band C). Therefore, the F-box proteins of some embodiments of the presentinvention display selectivity toward particular targets.

[0197] Recognition of Phosphorylated Cln1 and Cln2 by Grr1

[0198] Previous studies have shown that mutations of potential Cdc28phosphorylation sites in the C-terminal PEST domain in Cln2 increase itsstability in vivo (Lanker et al., Science 273:1597-1601 [1996]), andthat only the phosphorylated form of Cln2 is associated with Cdc53 invivo (Willems et al., [1996], supra), implicating this interaction inthe Cln destruction pathway. Cdc28 is required for Cln phosphorylationalthough it has not been determined that the requisite phosphorylationreflects autophosphorylation or phosphorylation by a distinct proteinkinase. The finding that Sic1 is recognized by the F-box protein Cdc4,together with a genetic requirement for the F-box protein Grr1 in Clndestruction, led to the next step in the development of the presentinvention, namely the examination of whether Grr1 functions inrecognition of phosphorylated Clns.

[0199] To generate Cln proteins for binding reactions, Cln/Gst-Cdc28complexes were isolated from insect cells. In the presence of ATP, bothCln1 and Cln2 were found to be autophosphorylated, a modification thatreduces their electrophoretic mobility (see below). To examine whetherGrr1 can associate with phosphorylated Clns and to compare the extent ofselectivity of Grr1 and Cdc4 toward Cln binding, anti-Skp1^(F) immunecomplexes from cells co-expressing Grr1 or Cdc4 in the presence orabsence of Cdc53 were used in binding reactions with ³²P-labeled Cln1 orCln2 kinase complexes. ³²P-labeled Sic1 was used as a control for Cdc4binding. Both Cln1 and Cln2 complexes were found to associate withGrr1/Skp1^(F)/Cdc53 complexes (See, FIG. 5A) with an efficiency of about40% of the input Cln1 or Cln2 (See, FIG. 5A, lanes 8 and 12) and thisassociation did not require Cdc53 (lane 16). In contrast, about 6% ofthe input Cln proteins associated with Cdc4/Skp1^(F) complexesindependent of the presence of Cdc53 (lanes 7, 11, and 15), comparedwith 1% association in the absence of an F-box protein (lanes 6, 10,14). The extent of selectivity of these F-box proteins for Cln and Sic1was further reflected by the observation that Cln1 protein present inthe phosphorylated Sic1 preparation was selectively enriched in Grr1complexes (FIG. 5A, lane 4). The presence of all proteins in the bindingreaction was confirmed by immunoblotting (FIG. 5B) and the quantities ofCdc4 and Grr1 were comparable, based on Coomassie staining of SDS gelsof immune complexes. Thus, Grr1 and Cdc4 display specificity towardphysiological substrates.

[0200] Cln1 Phosphorylation is Required for Recognition by Grr1

[0201] If Cln phosphorylation is required for ubiquitination assuggested by genetic studies (Lanker et al., [1996], supra; and Willemet al., [1996], supra), and if Grr1 is the receptor for Clns, then theGrr1/Cln interaction would be expected to be phosphorylation dependent.Thus, the next step in the development of the present invention was toexamine Grr1 alone and in complexes with Skp1 or Skp1/Cdc53. Thus, Grr1alone, or in complexes with Skp1 or Skp1/Cdc53 was immunoprecipitatedfrom insect cell lysates and used in binding assays with phosphorylatedor unphosphorylated Cln1 complexes (FIG. 5C).

[0202] Unphosphorylated Cln1 was produced in insect cells as a complexwith kinase deficient Gst-Cdc28(K−), which minimized Cln1autophosphorylation during expression and allowed the role ofphosphorylation to be tested. As isolated, this Cln1 protein migrated asa homogeneous species of approximately 66 kDa (FIG. 5C, lane 1). Incontrast, phosphorylated Cln1 (lane 2) undergoes a dramatic mobilityshift to approximately 80 kDa, consistent with the results observed invivo. Phosphorylated Cln1 (and its associated Cdc28 protein) efficientlyassociated with all Grr1 complexes (FIG. 5C, lanes 6, 8, 10), but wasabsent from control binding reactions lacking Grr1 (FIG. 5C, lane 4). Incontrast, the levels of unphosphorylated Cln1 associated with Grr1complexes were compared to that found in binding reactions lacking Grr1(FIG. 5C, lanes 3, 5, 7, 9). Thus, association of both Cln1 with Grr1and Sic1 with Cdc4 is greatly enhanced by phosphorylation. Although theGrr1/Skp1/Cdc53 complex is capable of binding efficiently tophosphorylated Cln1, it was not competent for Cln1 ubiquitination whensupplemented with Cdc34 and E1 (FIG. 5D). Moreover, Cdc4 complexes thatfunctioned in Sic1 ubiquitination also failed to catalyze ubiquitinationof Cln1 (FIG. 5D), despite the fact that Cln1 can associate, albeitweakly, with Cdc4 (FIG. 5A). In contrast, identical preparations ofphosphorylated Cln1 protein were efficiently ubiquitinated in partiallypurified yeast lysates in a Cdc34-dependent manner (See e.g, FIG. 5E),indicating that this preparation of Cln1 is competent forubiquitination. Although an understanding of the mechanism is notnecessary in order to use the present invention, the absence of Cln1ubiquitination in the purified system may reflect the requirement ofadditional factors or modifications.

[0203] F-box Proteins as Receptors for Ubiquitination Targets

[0204] The present invention contemplates that a large number ofproteins contain the F-box, and are thereby implicated in the ubiquitinpathway. The development of the present invention has revealed thatF-box proteins directly contact ubiquitination substrates and candisplay selectivity in recognition of potential targets forubiquitination, as would be expected of E3 proteins. For example, bothGrr1 and Cdc4 assemble into mutually exclusive complexes with Cdc53 andSkp1 (FIG. 4). However, Grr1 does not associate with Sic1, nor does itsupport Sic1 ubiquitination. In contrast, it was found that Cln proteinsefficiently associate with Grr1/Skp1^(F) complexes and withCdc4/Skp1^(F) (although less efficiently) (See e.g., FIG. 5). AlthoughCdc53 was originally isolated as a Cln2-interacting protein (Willems etal., [1996], supra), the present invention provides evidence that thisoriginal interaction was bridged by Grr1 and possibly Cdc4. The Grr1/Clninteraction is of interest in view of the fact that GRR1, CDC53, andSKP1 are required for destruction of Cln proteins, and suggests thatGrr1 functions as a component of an E3 for Cln ubiquitination. Theabsence of Cln ubiquitination by purified Grr1 complexes is likely toindicate the absence of an essential factor(s) or modifications that arenot required for Sic1 ubiquitination in vitro, and provides evidencethat Cln ubiquitination may be more complex than is Sic1 ubiquitination.Nonetheless, the present invention provides methods, compositions, andmodels for the development of compounds that interact with theubiquitination process, and thereby affect protein degradation throughany number of routes.

[0205] Despite the observation that F-box proteins may show selectivitytowards potential substrates, it is unlikely that F-box proteins will bemonospecific. For example, in S. pombe, recent genetic data have linkedthe CDC4 homolog pop + with the ubiquitination of both the CK1 Rum1 andCdc18, a regulator of DNA replication (Kominami and Toda, Genes Dev.,11:1548-1560 [1997]). In budding yeast, CDC4 has also been implicated indestruction of the Cdc18 homolog Cdc6 (Piatti et al., Genes Dev.,10:1516-1531 [1996]), indicating that it too has multiple targets. Itwas also determined that Cdc4 can associate with Clns, albeit lessefficiently than with Grr1 (FIG. 5). Of importance is the fact that allof the targets of F-box protein mediated destruction identified to dateare central regulators of key events in the cell, including DNAreplication, cell cycle progression, and nutritional sensing.

[0206] A Cdc53/Cdc4/Skp1 E3 Complex is Required for Sic1 Ubiquitinationby Cdc34

[0207] Sic1 destruction is genetically dependent upon Cdc34, Cdc4,Cdc53, and Skp1. During the development of the present invention, it wasdetermined that these proteins are directly involved in theubiquitination process. As Cdc53 can simultaneously bind the E2 Cdc34and Skp1, it frictions as an adapter linking the Skp1/F-box proteincomplex to E2s (FIG. 1). In turn, Skp1 has the ability to link Cdc4 toCdc53. Cdc4 binds both Skp1 and the ubiquitination substrate Sic1. Theinteraction of Cdc4 with Skp1 was shown to involve the F-box located inthe N-terminus of Cdc4, while the interaction with Sic1 involves Cdc4'sC-terminal WD-40 repeats (FIG. 2). Skp1 was also shown to be involved insubstrate recognition because it enhances the association of Cdc4 withphosphorylated Sic1. Cdc4 was shown to act as a receptor that, inconjunction with Skp1, recruits substrates to the ubiquitinationcomplex. It is contemplated that any of these proteins could also havecarrier roles in the transfer of ubiquitin like E6AP (See e.g.,Scheffner et al., Cell 75:495-505 [1995]). However, it was determinedthat mutation of the only conserved cysteine in Skp1 or all 6 cysteinesin Cdc53 did not impair complementation of skp1 or cdc53 null mutations,respectively, indicating that these two proteins are unlikely totransfer ubiquitin by a thio-ester intermediate.

[0208] Phosphorylation Directly Regulates Association of Sic1 and ClnProteins with E3s

[0209] A central feature in the recognition of Sic1 and Cln by F-boxproteins is the phosphorylation dependent nature of the interaction.Association of Sic1 with Cdc4-containing complexes and subsequentubiquitination requires Sic1 phosphorylation, as shown in FIGS. 2 and 3.It was also shown that Sic1 phosphorylated by excess Clb5/Cdc28 kinasecan be ubiquitinated in vitro (See, FIG. 3E). It is contemplated thatthe initial generation of Clb5/Cdc28 activity at the G1/S transitioncould potentially accelerate Sic1 destruction facilitating the sharp andunidirectional change of state characteristic of cell cycle transitions.

[0210] Similarly, association of Grr1 with Cln proteins is greatlyenhanced by phosphorylation, as indicated in FIG. 5. Phosphorylation ofspecific residues in the C-terminal PEST domain of Cln1 is required forCln2 instability (Lanker et al., [1996], supra), and phosphorylated Cln2is found in complexes with Cdc53 in vivo (Willems et al. [1996], supra).The present invention shows that Cln/Cdc28 can provide a system thatfunctions in vitro. The present invention also provides methods,compositions, and models for the determination of whether Clnubiquitination is activated by autophosphorylation in trans, as theaccumulation of active Cln/Cdc28 complexes may be required to achievesufficient Cln phosphorylation to promote its destruction.

[0211] While regulating the association of F-box proteins throughsubstrate phosphorylation is an effective method controlling the timingof ubiquitination, it is not necessarily the case that all F-boxproteins will recognize their substrates in a phosphorylation dependentmanner. Observations made during the development of the presentinvention indicate that WD-40 and LRR containing F-box proteins caninteract with phosphorylated substrates, but approximately half of theknown F-box proteins do not have obvious protein interaction motifs.Nonetheless, the present invention provides methods, compositions, andmodels to determine whether the interaction of these proteins with theirtargets is regulated by phosphorylation or even involves ubiquitination.The timing of ubiquitination could be controlled by mechanisms unrelatedto substrate phosphorylation, such as controlled accessibility ofsubstrates or regulated expression, localization, or modification of theF-box protein, thus providing methods for development of compounds thataffect proteolysis.

[0212] While the abundance of Cdc4 is not cell cycle regulated, theF-box protein Skp2 displays cell cycle-regulated mRNA abundance whichpeaks in S-phase, consistent with its association with cyclin A duringthat phase of the cycle (Zhang et al., [1995], supra). In vivo,association of Grr1 and Skp1 is enhanced in the presence of glucose in apost-translational mechanism.

[0213] A large number of proteins contain PEST sequences and in a subsetof these proteins, these sequences have been shown to be phosphorylatedand to mediate instability. The development of one embodiment of thepresent invention focused on the role of Skp1 and F-box proteins inassembly of a ubiquitination complexes that recognizes specificphosphorylated proteins. While the particular complex defined by thisembodiment of the present invention is unlikely to be responsible forrecognition of all PEST-dependent proteolysis substrates, this complexis likely to be the prototype for a diverse set of complexes in highereukaryotes. Five CDC53 homologs have been identified in mammals (Cul1-5;Kipreos et al., Cell 85:829-839 [1996]). approximately 15 E2-relatedgenes exist in S. cerevisiae alone, several dozen F-box containingproteins have been identified in several species, and several SKP1related genes exist in C elegans and are likely to exist in mammals aswell. It is clear that the present invention provides methods,compositions, and models to identify PEST-dependent proteolysissubstrates in these and other organisms, as well as providing theflexibility to differentially regulate the ubiquitination of a verylarge number of substrates.

[0214] Other Applications

[0215] In addition, various embodiments of the present invention finduse in other settings. For example, the methods, compositions, andmodels of the present invention provide the tools to determine thefunction of such proteins as elongin C, a Skp1-related protein is partof a complex containing the Cdc53-related protein Cul2, the vonHippel-Lindau (VHL) tumor suppressor protein, elongin B, and elongin A,a protein that is also found in association with elongin C, and containsan F-box. Thus, the present invention provides the means to developcompounds that affect systems other than ubiquitination-mediatedproteolysis.

[0216] Indeed, the F-box-directed ES complex (FEC) embodiment describedin detail herein, represents one example of a pathway through whichprotein kinases control the stability of target proteins. In view of thelarge number of protein kinases and possible FECs, this pathway may besecond only to transcriptional regulation in the control of proteinabundance. While the specific examples described herein focus on theconcern the cell cycle, the present invention provides methods,compositions and models applicable to other, diverse regulatory systems.

[0217] Although an understanding of the mechanism is not necessary inorder to use the present invention, FIG. 6A provides a model in which aprotein kinase phosphorylates target proteins, thus activating them forassociation with their receptors, the F-box proteins. Although someF-box proteins may already be associated with a Skp1/Cdc53 complex priorto association with substrates, as shown in FIG. 6A, it is also possiblethat F-box proteins exist in a unbound form, and that association of theF-box protein with the substrate drives association with Skp1/Cdc53.Since Skp1 enhances the association of Cdc4 with Sic1, depending on therelative Kd values for individual interactions and concentrations of theconstituents, association of the target with an F-box protein mayenhance association with Skp1. Once the ubiquitination complex is formedand polyubiquitination takes place with the assistance of E1 and E2proteins, the substrate is then released and recognized to the 26Sproteosome where it is proteolyzed.

[0218] As indicated in FIG. 6B, it is contemplated that othercombinations of FEC (or “SCF”) complexes exist in cells. For example,the F-box protein Met30 is closely related to Cdc4, and is required forrepression of genes in the methionine biosynthetic pathway in thepresence of S-adenosylmethionine (AdoMet) (See, Thomas et al., Mol Cell.Biol., 15:6526-6534 [1995]). Met30 forms a complex with Met4, atranscription factor required for methionine biosynthetic geneexpression. The present invention provides the means to determinewhether Met4 is ubiquitinated in response to adomethionine. Furthermore,although the primary embodiment of the present invention has focused onCdc34, the present invention provides means to determine whether otherE2s are capable of functioning in the context of FECs.

[0219] Also, as shown in FIG. 6C, SCF complexes (i.e., Skp1, Cdc53, andCdc4 present in a multiprotein complex), work together with proteinkinase signalling pathways to control protein abundance. FIG. 6Cillustrates one such pathway, in which SCF pathways function multipletimes in the transition from G1 to S phase in S. cerevisiae.

[0220] Like protein synthesis, protein destruction is a fundamentalmechanism used by organisms to manipulate their function. In oneembodiment, the present invention provides the composition of an E3complex, FEC, involved in selection of ubiquitination substrates.Because the constituents of this complex are members of proteinfamilies, the present invention provides the prototype for a large classof E3s formed by combinatorial interactions of related family members asindicated in FIG. 6B. The identification of F-box proteins as thereceptor components of this ubiquitin ligase provides the means foridentification of the key regulatory molecules controlled byubiquitin-mediated proteolysis. Thus, the present invention providesmeans for the elucidation of the biochemistry of this generalubiquitination pathway is likely to have important ramifications formany aspects of biology including cell proliferation, development, anddifferentiation.

[0221] The Present Invention in Action

[0222] The following example is provided to illustrate one specificapplication of the present invention. In this example, the methods andcompositions of the present invention are used to identify a novel E3ubiquitin ligase complex that finds use in such applications as theubiquitination of IκB, which has direct impact on the regulation ofNF-κB activity and associated cellular pathways. The findings of thesestudies provide new therapeutic targets for the NF-κB pathway that candiversify the existing programs for drug development.

[0223] The NF-κB pathway has many important physiological roles and hasbecome the focus of intense interest as a target for drug development.For example, the NF-κB pathway has been implicated in regulation ofapoptosis. Hallmarks of transformed cells include the ability toproliferate with reduced growth factor levels and defects in the abilityto undergo apoptosis. Many cell types contain signaling systems thatrecognize inappropriate proliferation and respond by activating anintrinsic apoptotic pathway leading to cell loss. For example, it hasbeen shown that loss of the Cdk inhibitor p57 in the lens leads to bothinappropriate proliferation and increased apoptosis (Zhang et al.,Nature 387:151 [1997]). As such, transformation pathways frequentlyinclude some process that either inactivates a component of theapoptotic machinery, activates a survival pathway, or both. TNF-α, apro-inflanmmatory cytokine, functions in part to activate NF-κB, atranscription factor composed of p50 and p65/Rel subunits (Baeuerle andBaltimore, Cell 87:13 [1996]; Beg et al., Mol. Cell. Biol. 13:3301[1993]; DiDonato et al., Mol. Cell. Biol. 15:1302 [1995]; and Tewari andDixit, Genes & Devel. 6:39 [1996]). NF-κB also activates the expressionof a large number of genes, including growth factors, chemokines, andadhesion molecules which mediate inflammatory responses.

[0224] TNF-α has also been shown to induce particular cell types toundergo apoptosis, although the cytotoxic effects are revealed mostfrequently only if protein/RNA synthesis is blocked (Tewari and Dixit,supra). Recent studies have revealed that the inability of cells toundergo apoptosis in response to TNF-α reflects activation of a survivalpathway, which is programmed by NF-κB action (Beg and Baltimore, Science274:782 [1996]; Liu et al., Cell 87:565 [1996]; Van Antwerp et al.,Science 274:787 [1996]; and Wang et al., Science 274:784 [1996]). Cellslacking RelA or blocked for NF-κB nuclear translocation are sensitive toTNF-mediated killing (Beg and Baltimore [1996], supra; and Wang et al.,supra). Moreover, induction of NF-κB activity protects cells againstTNF-mediated cell death (Van Antwerp et al., supra). TNF-α may inducecell death through one pathway and simultaneously induce a protectivemechanism through NF-κB (Beg and Baltimore [1996], supra). These studiesindicated for the first time an important role for NF-κB in cellsurvival pathways and suggested inhibition of NF-κB function might beused to predispose cancer cells to killing by TNF-α or chemotherapeuticcompounds.

[0225] In principle, agents that block NF-κB function could inactivatethe cell survival pathway set in motion by NF-κB, rendering cellscapable of undergoing apoptosis. In addition to its survival functions,there is evidence that NF-κB may play growth promoting roles byactivating transcription of myc, which may drive the cell cycle forward(reviewed by Sovak et al., J. Clin. Invest. 100:2952 [1997]). There isaccumulating evidence that NF-κB is used to set up a survival pathway intransformed mammary cells. Activated nuclear NF-κB is prominent inmammary tumor lines (Nakshatri et al., Mol. Cell. Biol. 17:3629 [1997]),but rare in normal mammary epithelial cells, and recent studies indicateblocking NF-κB in this setting can induce apoptosis (Sovak et al.,supra). Other cell types such as B-cells also undergo apoptosis whenNF-κB is inhibited (Wu et al., EMBO J. 15:4682 [1996]). It is possiblethat NF-κB is normally used to protect particular mammary cells fromapoptosis, which is occurring as part of the normal biology of thesystem, and that transformation takes advantage of this property. Inaddition, NF-κB activation in mammary tumor cells correlates withER-independent proliferation (Nakshatri et al., supra), suggesting apossible link between estrogen responsiveness and apoptosis.

[0226] For the last several years, there has been interest in the drugsthat block NF-κB activation for use in anti-inflammatory diseases (Seee.g., Vogel, Science 281:1943 [1998]), an interest that has beenstrengthened by the finding that aspirin functions to block the NF-κBpathway (Grilli et al., Science 274:1383 [1996]). The finding that NF-κBalso functions in cell survival has led to the realization that drugsthat affect this pathway may also be useful in cancer treatment. Theinsensitivity of some tumor cells to chemotherapeutics may reflect aninability to undergo apoptosis and interestingly, inhibitors of NF-κBcan correct the radiation sensitivity of cells mutant in the AT gene(Jung et al., Science 268:1619 [1995]). Thus, NF-κB inhibitors may finduse as an adjunct to chemotherapy/radiotherapy. Thus, it is contemplatedthat a more complete understanding of the NF-κB activation pathway willlead to the identification of new therapeutic targets.

[0227] NF-κB activation involves a multi-step signal transductionpathway (Baeuerle and Baltimore, supra) involving receptor activation,activation of kinases (IKKα and IKKβ) that phosphorylate IκB (theendogenous inhibitor of NF-κB), ubiquitination of IκB, proteolysis ofIκB, and translocation of NF-κB to the nucleus. Recent advances includeidentification of IKKs (DiDonato et al., Nature 388:548 [1997]; Mercurioet al., Science 278:860 [1997]; Regnier et al., Cell 90:373 [1997];Woronicz et al., Science 278:866 [1997]; and Zandi et al., Nature387:151 [1997]) and the components of the TNF receptor complex (reviewedby Tewari and Dixit, supra). In contrast, prior to the presentinvention, virtually nothing was known about the molecules that functionin the ubiquitination step.

[0228] As discussed above, the present invention provides a novel E3ubiquitin ligase complex that provides means to identify therapeutictargets for regulating NF-κB activity, to identify the moleculardeterminants that confer the ability of this ligase to recognizephosphorylated IκB, and to identify molecules that can disrupt thisinteraction.

[0229] A. Background Regulation of NF-κB Function

[0230] NF-κB activity is regulated primarily through its sub-cellularlocalization (Baeuerle and Baltimore, supra). In the absence of signal,NF-κB is sequestered in the cytoplasm by interaction with a member ofthe IκB (inhibitor of κB) family of proteins (Baeuerle and Baltimore,Science 242:540 [1988]). IκB binds to p50/p65 heterodimers andsimultaneously blocks both the nuclear localization signal and theability of NF-κB to bind DNA (Beg et al., Genes & Devel. 6:1899 [1992];Luque and Gelinas, Mol. Cell. Biol. 18:1213 [1998]; and Thompson et al.,Cell 80:573 [1995]). In response to stimuli intended to activate NF-κB,IκB is rapidly phosphorylated (Beg et al., Mol. Cell. Biol. 13:3301[1993]; Brown et al., Science 267:1485 [1995]; Chen et al., Genes &Devel. 9:1586 [1995]; DiDonato et al., [1995], supra; Finco et al.,Proc. Natl. Acad. Sci. 91:11884 [1994]; Lin et al., Proc. Natl. Acad.Sci. 92:552 [1995]; and Liu et al., Cell 87:565 [1996]). This signalsIκB to be destroyed by ubiquitin mediated proteolysis, allowing NF-κB totranslocate to the nucleus to activate target genes (Alkalay et al.,Proc. Natl. Acad. Sci. 92:10599; Henkel et al., Nature 365:182 [1993];and Scherer et al., Proc. Natl. Acad. Sci. 92:11259 [1995]). Theidentity and regulation of the ubiquitin ligase that functions in NF-κBubiquitination was unknown in the art.

[0231] A key component of this signaling pathway involves activation ofkinases responsible for IκB phosphorylation, since this step (i.e., IκBphosphorylation) is thought to be the rate-limiting step in NF-κBactivation. Signaling molecules such as TNF, which promoter NF-κBactivation in particular cell types, bind to TNF receptors that link tothe death domain protein TRADD, and TRAF1/2 which contain a TRAF domain(Tewari and Dixit, supra). These proteins function in the transientactivation of two kinases, IKKα and IKKβ, which are part of a large (700kd) complex whose other components are not yet fully defined (DiDonatoet al., [1997], supra; Mercurio et al., supra; Regnier et al., Cell90:373 [1997]; Woronicz et al., Science 278:866 [1997]; and Zandi etal., supra). In vitro, each of these kinases specifically phosphorylateIκB on two serine residues (Ser-32 and Ser-36) and this combination ofphospho-serine residues is thought to be the targeting signal for IκBubiquitination, although how this signal is recognized and utilized isnot known in the art Importantly, overexpression of non-phosphorylatableIκB has been shown to be effective in blocking NF-κB activation(Woronicz et al., supra).

[0232] B. Phosphorylation Specific Association of IκB with Skp1

[0233] The role of SCF complexes in phosphorylation-dependentubiquitination led to the examination of whether IκB might associatewith Skp1. HeLa cell lysates were incubated with agarose beads (Affigelbeads) containing unphosphorylated and phosphorylated IκB sequencesoverlapping the ubiquitination targeting signal previously identified inIκB (Yaron et al., EMBO J. 16:6486 [1997]; containing Ser-32 and Ser-36)and the presence of Skp1 in IκB-associated proteins examined byimmunoblotting, as shown in FIG. 8. The results demonstrated that Skp1specifically associated with phosphorylated IκB but not unphosphorylatedIκB. Skp1 is a highly abundant protein and is thought to be distributedamong multiple F-box proteins and possibly kinetochore complexes. It isestimated that ˜1% of the Skp1 in these extracts can associate with IκBin vitro.

[0234] C. Slimb F-Box Protein Associates with Phosphorylated IκB

[0235] Having found that Skp1 can associate with phosphorylated IκB, anumber of F-box proteins were surveyed for association withphosphorylated IκB. Various F-box proteins were produced by in vitrotranslation and tested for binding to phospho-IκB and IκB. Inparticular, a variety of in vitro translated F-box proteins containingLRRs (Skp2, F alpha), WD40 repeats (MD6, Met30), a cyclin box (cyclinF), and no obvious additional domains (F gamma) failed to interact withphosphorylated IκB, as shown in FIG. 9. In contrast, the slimb protein(also referred to herein as “TRCP protein”) specifically associated withphosphorylated IκB, suggesting that slimb F-box protein plays a role inIκB/NF-κB regulation.

[0236] To determine cell types where slimb might function, asystematicin situ hybridization analysis was initiated to determine patterns ofslimb expression in adult mouse tissues and during development. Asection through a E12.5 day mouse was subjected to in situ hybridization³⁵S-labeled mouse slimb antisense RNA using established procedures(Zhang et al., supra). Analysis demonstrated that slimb is expressed atmaximal levels in the ventricles of the forebrain and hindbrain, lung,and liver. Weaker expression was observed throughout most of the embryo.

[0237] D. slimb/Skp1 Associates with Phosphorylated IκB

[0238] The finding that Skp1 and slimb can both form complexes withphosphorylated IκB beads, together with the fact that slimb contains anF-box, led to the examination of whether slimb can associate with Skp1and Cul1 in vivo. Although every F-box protein tested to date interactswith Skp1, there are 6 Cul homologs and it is not clear at presentwhether they all bind to Skp1 or only a subset bind to Skp1. To examinethese interactions, 293 T-cells were transfected with vectors expressingvarious tagged versions of Skp1, slimb, and cul1 as shown in FIGS. 10Aand 10B. To assemble the SCF/slimb complex, plasmids expressing cul1HA,Skp1HA3, and slimbMYC9 were transfected in the indicated combinations(FIG. 10A) into 293 cells using lipofection. After 48 hours, cells weredisrupted in lysis buffer (10 mM Tris-HCl, 0.5% nonidet P-40, 150 mMNaCl, 10 mM beta-glycerolphosphate) and insoluble material removed bycentrifugation. Lysates (1 mg of protein) were subjected toimmunoprecipitation using anti-myc antibodies. Immune complexes werewashed three times in lysis buffer and were separated by SDS-PAGE andtransferred to nitrocellulose. Blots were developed using anti-HA,anti-Skp1, and anti-myc antibodies. FIG. 10B shows lysates from theindicated transfections that were subjected to immunoblotting using theindicated antibodies.

[0239] Additionally immunoprecipitation/Western blotting experimentswere performed as shown in FIGS. 11A and 11B. In FIG. 11A, the indicatedplasmids were transfected into 293T cells and after 48 hours, lysateswere made and subjected to immunoprecipitation using anti-HA antibodiesto precipitate Cul1. The presence of slimb and Skp1 were determinedusing anti-myc and anti-Skp1 antibodies. The myc9-tagged Skp1 migratesat approximately 30 kilodaltons compared to 19 kd for untagged Skp1.

[0240] The data in FIGS. 10 and 11 demonstrate that Cul1 immunocomplexescontain Skp1, as expected, but also contain slimb. Likewise, althoughthe data are not included herein, it was shown that slimbimmunocomplexes contain Skp1 and Cul1. To examine whether slimb/Skp1complexes are capable of associating with phosphorylated IκB, 293T-cells were transfected with CMV-HA slimb and CMV-HA Skp1 and lysatessubsequently incubated with IκB or phosphorylated IκB beads prior toSDS-PAGE and Western analysis with anti-HA antibodies as shown in FIG.11B. In this Figure, lysates from the indicated transfected cells weresubjected to binding reactions using immobilized IκB or phospho-IκB.After washing, bound proteins were subjected to immunoblotting withanti-HA to visualize slimb and Skp1 proteins. As shown, Skp1 and slimbassociate specifically with phospho-IκB (i.e., transfected slimb andSkp1 assemble into complexes that are recognized by phospho-IκB).

[0241] HA slimb was found to associate with phosphorylated but notunphosphorylated IκB beads with or without transfection of Skp1. HA Skp1also associated with IκB in a phosphorylation-specific manner. Uponlonger exposure of this blot, HA Skp1 was detectable in complexes withphosphorylated (but not unphosphorylated) IκB in lysates from cellstransfected with HA Skp1 alone, suggesting that HA Skp1 can assemblewith the endogenous slimb protein. Previous studies indicated that apeptide containing the sequence KKERLLDDRHDSGLDSMKDEE (residues 21-41from IκB; SEQ ID NO:60) will not inhibit IκB ubiquitination when addedin vitro to a crude cell lysate which supports IκB ubiquitination in amanner that is dependent upon the phosphorylation of Ser-32 and Ser-36in IκB. In contrast, the same peptide that has been phosphorylated onSer-32 and Ser-36 will block the ubiquitination of IκB. Similarly,phosphorylated IκB peptide will block nuclear translocation of NF-κB inintact cells in response to stimuli while the unphosphorylated peptidewill not. It is known that IκB needs to be phosphorylated on these twoserines by IKK for ubiquitination to occur and this phosphorylationserves as the signal. These phosphopeptides derived from IκB are thoughtto block IκB ubiquitination by competing with the full-length IκBsubstrate for the recognition factor of the ubiquitin ligase that isnormally functioning in IκB ubiquitination. Thus, the finding of thepresent invention that this same phosphorylated IκB peptide, but not theunphosphorylated peptide, will specifically interact with the SCF slimbcomplex suggests that this slimb complex is the ubiquitin ligase forIκB. Thus, the present invention provides a novel E3 ubiquitin ligasecomplex, thereby providing means to identify therapeutic targets forregulating NF-κB activity, to identify the molecular determinants thatconfer the ability of this ligase to recognize phosphorylated IκB, andto identify molecules that can disrupt this interaction.

[0242] These studies have revealed that slimb recognizes thephosphorylated targeting signal in IκB. It is contemplated that othercellular or viral proteins contain these sequences and will be thereforetargeted to the slimb ubiquitin ligase. Although this sequence isrecognized by slimb, it is further contemplated that other unrelatedsequences may also interact with slimb possibly through independentdomains. It is also contemplated that other F-box proteins containinganalogous mutations will find use to demonstrate the specificity of thedominant negative effect.

[0243] E. Further Characterization

[0244] Using the methods and compositions of the present invention,there are several approaches available to further characterize therelationship between the SCF slimb complex and IκB. These include bothin vivo and in vitro approaches.

[0245] In vivo: In one embodiment of the present invention, NF-κBactivation or IκB destruction is blocked using a dominant negative formof slimb. A dominant negative form of slimb is one that will still bindto IκB but will not assemble with the cul1/Skp1 complex. Therefore, thedominant negative slimb protein, when expressed at sufficient levels intransfected cells, would bind phosphorylated IκB, thereby blockingaccess of the endogenous slimb protein to IκB. Since this dominantnegative IκB is not assembled with cul1/Skp1 complexes, appropriate E2conjugating enzymes would not be physically coupled to IκB and wouldtherefore not carry out the ubiquitination reaction. Many forms of slimbfind use as dominant negative proteins and are made using methodsstandard in the art. For example, in preferred embodiments, versions ofslimb that either lack the F-box domain or contain one or more pointmutations in the F-box domain are used. This domain is required forinteraction with Skp1, and mutation of the F-box in the appropriateresidues blocks association with Skp1. The preferred residues to beuseful in this regard include those that are highly conserved in otherF-boxes. Association with Skp1 in vitro could be used to demonstratethat the mutant slimb protein no longer interacts with Skp1. Thefunction of the slimb dominant negative protein is assessed, forexample, by monitoring NF-κB activity on a reporter construct, thetranslocation of NF-κB to the nucleus in response to TNF treatment, orstabilization of IκB protein levels.

[0246] In vitro: In one embodiment of the present invention, the rate ofIκB ubiquitination in cells is directly altered by blocking oractivating slimb function. For example, in one series of experiments,the ubiquitination of IκB is blocked using slimb mutants (i.e., dominantnegative F-box mutants) that bind IκB but not Skp1, thereby uncouplingIκB's ability to associate with endogenous SCF slimb when the mutant isoverexpression. A set of conserved residues in the F-box whose mutationabolishes interaction of the F-box protein Cdc4 with Skp1 has previouslybeen identified (Bai et al., Cell 86:263 [1996]). In one set ofexperiments, two sets of conserved F-box residues (LP and IL) in slimbare mutated to AA and act to verify binding to phospho-IκB but not Skp1in vitro. Appropriate mutants are transfected into HeLa cells and theeffects on TNF-induced activation of NF-κB is assessed using threeprimary assays: 1) pulse chase analysis of IκB (when a high level oftransfection is achieved), 2) NF-κB activated reporter (e.g.,luciferase) activity, and/or 3) entry of Rel into the nucleus byimmunofluorescence. Other F-box proteins (including the WD40 containingMD6), mutant in the F-box, are used as controls.

[0247] The results of experiments conducted during the development ofthe present invention indicate that slimb levels are low compared to thelevels of transfected slimb. Thus, in preferred embodiments of thepresent invention, the dominant approach is used. For confirmation or asalternative embodiments, other approaches such as antisense are used.For example, the antisense approach has been used to successfully blockIKK activity (DiDonato et al., [1997], supra).

[0248] In yet other embodiments of the present invention, the role ofslimb in the ubiquitination of IκB is characterized. In one embodiment,overexpression of SCF slimb components is used to enhance theunstimulated rate of IκB ubiquitination.

[0249] In other embodiments, the activity of the SCF slimb complextoward IκB is demonstrated in vitro. For example, experiments conductedduring the development of the present invention have demonstrated thatcells can be transfected with slimb, cul1, and Skp1 to generatecomplexes. In the in vitro embodiments, experiments are conducted toexamine whether slimb alone or in combination with Skp1 and Cul1accelerates ubiquitination of endogenous or co-transfected IκB, usingpulse chase analysis or direct ubiquitination assays. Overexpression ofIκB increases its levels such that the endogenous slimb complex does notefficiently ubiquitinate it, thereby providing a window for accelerationby exogenous slimb. Direct ubiquitination analysis is achieved byco-transfection of a tagged ubiquitin plasmid followed byimmunoprecipitation of IκB and immunoblotting for the tagged ubiquitin.

[0250] In yet other embodiments of the present invention, methods todetermine whether slimb transfection can force NF-κB activation in theabsence of stimulation as a result of residual IKK activity, or withreduced levels of stimuli are conducted. Controls include slimb mutantsthat cannot bind IκB, and F-box proteins that do not associate with IκB(as described above).

[0251] As described above, the present invention provides approaches forreconstruction the SCFCdc4 ubiquitin ligase pathway for the Cdkinhibitor Sic1 (See also, Skowyra et al., Cell 91:209 [1997]). Thisapproach also finds use with slimb. First, in some embodiments,experiments are conducted to determine whether slimb immune complexesfrom transfected cells contain IκB ubiquitin ligase activity usingphosphorylated IκB or IκB point mutants in phosphorylation sites assubstrates. Cdc34 is the most likely candidate for the E2, however otherE2 are also tested (e.g., Ubc4, 5, and 10). The development of thissystem provides a screening assay to examine whether particularmolecules function to block IκB ubiquitination.

[0252] In yet other embodiments of the present invention, methods areprovided to determine whether interference with slimb inhibits NF-κBfunction and induces apoptosis. There is clear evidence that blockingNF-κB action in mammary tumor cells (578T) (Sovak et al., supra) and inother cell types (Van Antwerp et al., supra; Wang et al., Science274:784 [1996]; and Wu et al., supra) can lead to apoptosis. In one setof experiments, synthetic peptides overlapping the IκB recognitionsequence (in either the phosphorylated or unphosphorylated forms) aregenerated and microinjected (as described by Connell-Crowley et al.,Curr. Biol. 8:65 [1997]) into 578T human mammary tumor cells. Apoptosis,as well as the fate of NF-κB/IκB, is assessed by TUNEL andimmunofluorescence, respectively, using standard methods. In otherembodiments methods are provided to test whether dominant negative slimbor appropriate controls will induce apoptosis.

[0253] F. Slimb/IκB Interaction Surfaces as a Target for Drug Design

[0254] Due to the widespread interest in the generation of anti-NF-κBtherapeutics, many steps in the NF-κB pathway are being targeted.Because the nature of the ubiquitin ligase for IκB was unknown in theart, prior to the present invention, this step had not yet beenexplored. The slimb complex of the present invention provides a noveltarget and provide means to identify anti-NF-κB therapeutics Forexample, one major advantage of slimb is that it recognizes a smallphosphopeptide sequence. It is contemplated that molecules that mimicthis phosphopeptide and block NF-κB activation will be identified usingthe method of the present invention.

[0255] In some embodiments, the first steps in generating slimb/IκBinteraction surfaces involve identification of the molecular interactionsurfaces (interacting motifs) between slimb and IκB, and identificationof peptides or proteins that, by virtue of binding to slimb, blockbinding to IκB. These steps identify and provide motifs and assays thatfind use in screening combinatorial libraries for small moleculeinhibitors of the interaction. As there are many alternative approachesthat could be taken to identify molecular interaction surfaces, it isnot intended that the present invention be limited to any specificapproach. Preferred approaches are illustrated below, although thepresent invention is not limited to these particular approaches.

[0256] In a first embodiment of the present invention, a modifiedversion of the reverse two-hybrid approach (See e.g., Vidal et al.,Proc. Natl. Acad. Sci. 93:10315 [1996]) is applied to identify pointmutants in slimb that abolish IκB binding. This approach uses the powerof genetics to screen a large library of point mutants in slimb (e.g.,generated by either chemical mutagenesis or PCR using standard methods)to identify those that have lost the ability to bind to a target. Slimbmutants that fail to interact with IκB will be counter-screened forinteraction with Skp1 and for expression of full-length slimb mutantprotein using methods similar to those illustrated in FIG. 12. In thisFigure, phosphorylation-specific interaction of SCF slimb complexes withIκB peptide sequences were analyzed. Lysates (1 mg) prepared asdescribed above for FIG. 10 were incubated with 10 microliters ofaffigel beads containing either the IκB peptide or the same peptidecontaining phosphoserine at both serine residues. Beads were washedthree times with lysis buffer and bound proteins separated by SDS-PAGE.Proteins were transferred to nitrocellulose and used for immunoblottingwith the indicated antibodies. A subset of slimb mutants (determined bysequencing) that pass the secondary tests likely reside in IκB contactsequences.

[0257] Preferred interaction surfaces for use in screening assays arethose that have clustered mutations (e.g., those that are localizednearby on the same surface). To ensure that mutations reflect aninteraction site as opposed to structural alterations, the samples areassayed for second-site revertants in IκB that regenerate interactionwith a mutant slimb protein. These studies, together with conventionaldeletion analysis provide information about the necessary and sufficientsequences in slimb.

[0258] In other embodiments of the present invention, consensussequences are determined for interaction with the phosphopeptide bindingsite(s) in slimb. The small size and simplicity of the IκB sequencemakes it an attractive candidate for determining a consensus bindingsequence. In one embodiment of the present invention, a peptide libraryapproach is used to identify consensus sequences for phosphopeptiderecognition. The technique (See e.g., Songyang et al., Cell 72:767[1993]; Songyang et al., Mol. Cell. Biol. 14:2777 [1994]; and Songyanget al., J. Biol. Chem. 270:14863 [19951]) involves applying a highlycomplex mixture of peptide sequences that contain phosphoserines threeresidues apart (as in IκB), but are otherwise degenerate, to immobilizedslimb or the minimal interaction domain identified above. After thecolumn is washed, peptides are eluted and sequenced to determineconsensus sequences. Individual sequences are then tested for binding.The goal here is to define how selective the interaction site is. It isknown for instance that the spacing between the phosphoserines isrequired for IκB to be destroyed (Yaron et al., supra). The elucidationof such a consensus provides a theoretical “sequence space” and astarting point for drug discovery. It is also contemplated that thismotif will find use to search databases for other potential slimbsubstrates and/or regulators. In alternate embodiments, a particularpeptide sequence in the context of two glutamic acids (which can mimicphosphoserine) may be able to associate with slimb. In yet Rotherembodiments, peptide library experiments are performed with fixedglutamic acids to determine if any sequences exist that compete in aphosphorylation-independent manner. This provides a starting point fornon-phosphorylatable slimb inhibitors. To determine whether the peptidesidentified inactivate slimb in cells, peptides are microinjected intotissue culture cells and NF-κB function, as well as apoptosis, in 578Tcells is determined (as described above).

[0259] In other embodiments of the present invention, an alternativeapproach to the directed search for competitive binding components isused, which combines the complex nature of the human genome or peptideaptomer libraries coupled with the power of the reverse two hybridapproach. In this embodiment, cDNA or peptide aptomer libraries are betransformed into yeast strains expressing GAL4-IκKB and ACT-slimb andcells are selected for the loss of the IκB/slimb interaction. Libraryplasmids are rescued and sequenced to identify binding components, withfurther analysis revealing whether these proteins/peptides disrupt theinteraction by binding to one or both of the proteins. Peptide aptomersare then assessed as described above for synthetic peptides.

[0260] From these illustrative examples, it is clear that the presentinvention provides means to develop anti-NF-κB therapies based on theblocking of IκB ubiquitination. More generally, the identification andcharacterization of slimb as a member of an SCF complex illustrates thatthe methods and compositions of the present invention are capable ofidentifying and isolating F-box proteins and detecting F-box proteintargets and F-box protein complexes.

EXPERIMENTAL

[0261] The following examples are provided in order to demonstrate andfurther illustrate certain preferred embodiments and aspects of thepresent invention, and are not to be construed as limiting the scopethereof.

[0262] In the experimental disclosure which follows, the followingabbreviations apply: h (human); Sc (Saccharomyces cerevisiae); m(mouse); Ub (ubiquitin); E1 (Ub activating enzyme); E2 (Ub carrierprotein); E3 (Ub-protein ligase); ° C. (degrees Centigrade); rpm(revolutions per minute); BSA (bovine serum albumin); CFA (completeFreund's adjuvant); IFA (incomplete Freund's adjuvant); IgG(immunoglobulin G); IM (intramuscular); IP (intraperitoneal); IV(intravenous or intravascular); Sc (subcutaneous); H₂0 (water); HCl(hydrochloric acid); aa (amino acid); bp (base pair); kb (kilobasepair); kd (kilodaltons); gm (grams); μg (micrograms); mg (milligrams);ng (nanograms); μl (microliters); ml (milliliters); mm (millimeters); nm(nanometers); μm (micrometers); M (molar); mM (millimolar); MW(molecular weight); sec(s) (second/seconds); min(s) (minute/minutes);hr(s) (hour/hours); MgCl₂ (magnesium chloride); NaCl (sodium chloride);DTT (dithiothreitol); OD₂₈₀ (optical density at 280 nm); OD₆₀₀ (opticaldensity at 600 nm); PAGE (polyacrylamide gel electrophoresis); PBS(phosphate buffered saline [150 mM NaCl, 10 mM sodium phosphate buffer,pH 7.2]); PEG (polyethylene glycol); PMSF (phenylmethylsulfonylfluoride); SDS (sodium dodecyl sulfate); SDS-PAGE (sodium dodecylsulfate polyacrylamide gel electrophoresis); LMA (low meltingtemperature agarose gel; Tris (tris(hydroxymethyl)aminomethane); NETN(20 mM Tris-HCl, pH 8, 100 mM NaCl, 1 mM EDTA, 0.5% NP-40, 5 mM NaF, 30mM p-nitrophenylphosphate, 1 μg/ml each leupeptin and antipain, and 1 mMPMSF); TBST (20 mM Tris (pH 8), 100 mM NaCl, 0.5% Tween-20); IPTG(isopropyl-β-D-thiogalactopyranoside); LB (Luria-Bertani medium; perliter: 10 g tryptone, 5 g yeast extract, 10 g NaCl, pH 7; sterilized byautoclaving for 20 minutes at 15 lbs/in²); vol (volume); w/v (weight tovolume); V/V (volume to volume); Amersham (Amersham Life Science, Inc.,Arlington Heights, Ill.); ICN (ICN Pharmaceuticals, Inc., Costa Mesa,Calif.); Amicon (Amicon, Inc., Beverly, Mass.); ATCC (American TypeCulture Collection, Rockville, Md.); Becton Dickinson (Becton DickinsonLabware, Lincoln Park, N.J.); BioRad (BioRad, Richmond, Calif.);Clontech (CLONTECH Laboratories, Palo Alto, Calif.); Difco (DifcoLaboratories, Detroit, Mich.); GIBCO BRL or Gibco BRL (LifeTechnologies, Inc., Gaithersburg, Md.); Babco (Berkeley AntibodyCompany, Richmond, Calif.); Invitrogen (Invitrogen Corp., San Diego,Calif.); Kodak (Eastman Kodak Co., New Haven, Conn.); New EnglandBiolabs (New England Biolabs, Inc., Beverly, Mass.); Novagen (Novagen,Inc., Madison, Wis.); Qiagen (Chatsworth, Calif.); Pharmacia (Pharmacia,Inc., Piscataway, N.J.); Sigma (Sigma Chemical Co., St. Louis, Mo.);Sorvall (Sorvall Instruments, a subsidiary of DuPont Co., BiotechnologySystems, Wilmington, Del.); Stratagene (Stratagene Cloning Systems, LaJolla, Calif.); Whatman (Whatman LabSales, Hillsboro, Oreg.); BethylLaboratories (Bethyl Laboratories, Montgomery, Tex.); and Zeiss (CarlZeiss, Inc., Thornwood, N.Y.).

[0263] Unless otherwise indicated, all restriction enzymes were obtainedfrom New England BioLabs and were used according to the manufacturer'sinstructions; all oligonucleotide primers, adapter and linkers weresynthesized using standard methodologies on an ABI DNA synthesizer. Allchemicals were obtained from Sigma unless otherwise indicated.

EXAMPLE 1 Preparation of Antibodies

[0264] In this Example, anti-Skp1 and anti-Sic1 antibodies wereprepared. Using standard methods as known in the art, anti-Skp1 andanti-Sic1 polyclonal antibodies were generated in rabbits, withbacterial Gst fusion protein described below, used as the antigen.

[0265] A. Antigen Preparation

[0266] Expression plasmids for GST-SKP1 and GST-SIC1 were generated byligating open reading frames for the encoded proteins into pGEX2TK(Pharmacia), using established procedures known in the art (See e.g., J.Sambrook et al, supra). The Genbank accession numbers for SKP1 and SIC1are U61764 and X78309, respectively.

[0267] Plasmids were transformed into E. coli strain BL21(DE3)(Novagen). For expression, 1 L of E. coli cells were grown in LBmedium containing 0.1 mg/ml ampicillin at 37° C., until the OD₆₀₀reached 0.8. Expression was induced with 400 mM IPTG for three hours.Cells were harvested by centrifugation (2,000×g for 10 minutes), andthen lysed in 70 ml NETN buffer for 30 minutes, on ice. The insolublematerial was then removed by centrifugation (14,000×g, for 20 minutes).The lysate was then incubated with 0.5 ml glutathione Sepharose(Pharmacia) for 1 hour at 4° C. The Sepharose beads were washed threetimes with 10 ml NETN buffer, and washed twice with 5 ml of 100 mM NaCl,and the protein was eluted with buffer containing 0.5 ml 100 mM Tris (pH7.5), 100 mM NaCl, 40 mM glutathione. The protein was then stored at−80° C., prior to its use in the affinity purification of antibodies.

[0268] B. Antibody Production and Affinity Purification

[0269] Polyclonal rabbit anti-Cdc34 and anti-Cdc4 sera (provided by M.Goebl), as well as anti-Sic1, were affinity purified using recombinantantigens immobilized on nitrocellulose. The anti-Skp1 antibodies werenot affinity purified.

[0270] To affinity purify the anti-Sic1 antibodies, GST-Sic1 protein(0.1 mg) was subjected to electrophoresis on a 12% polyacrylamide(SDS-PAGE) gel, the protein was blotted to nitrocellulose (3 hours, at350 mA). Nitrocellulose filters containing GST-Sic1 protein wereincubated with 1 ml of anti-Sic1 antibodies for 3 hours, the filterswere washed twice with 10 ml of buffer containing 50 mM Tris (pH 7.5),50 mM NaCl, 0.5% Tween-20, and then eluted with 1 ml of 100 mM glycine(pH 2), and stored at 4° C. until use.

[0271] In addition to the anti-Skp1 and anti-Sic1 polyclonal rabbitantibodies generated in this Example, and the anti-Cdc34 and anti-Cdc4polyclonal rabbit antibodies from Dr. Goebl, monoclonal antibodies werealso used in the following Examples. These commercially availablemonoclonal antibodies were obtained from Babco (anti-HA, anti-Myc),Novagen (anti-T7 gene10, [i.e., “G10”]), and Kodak (anti-Flag, M2).

EXAMPLE 2 Expression, Purification and Phosphorylation of RecombinantProteins

[0272] In this Example, recombinant proteins were expressed, purifiedand phosphorylated. In these experiments, insect cells and baculoviruseswere used. Baculovirus expression vectors were generated in this Exampleusing the vectors in combination with linearized BaculoGold or AcMNPVwild-type DNA (Pharmingen). The viruses, their tags, and base vectorsare listed in Table 1.

[0273] Cdc4ΔWD is a mutant version of Cdc4 that contains a stop codon atresidue 566, which removes the last three WD-40 repeats. Gst-Cdc28HA(D154N), also referred to as “Gst-Cdc28HA(K−),” is a kinase-impairedform of Cdc28. In complexes with either Cln1 or Clb5, this kinase wasfound to exhibit <2% activity toward histone H1.

[0274] For expression of His₆Cdc34 and His₆-Sic1, plasmids weretransformed into BL21 (DE3) cells (Novagen). One liter of cells weregrown in LB containing 0.1 mg/ml ampicillin, at 37° C., until an OD₆₀₀of 0.8 was reached. Expression was then induced with 400 mM IPTG forthree hours. Cells were harvested by centrifugation (2,000×g, for 10minutes), lysed in 70 ml of 20 mM sodium phosphate buffer (pH 7.5)containing 500 mM NaCl, and 0.1 mg/ml lysozyme (Sigma), and incubatedfor 45 minutes on ice. Insoluble material was removed by centrifugation(14,000×g, for 20 minutes). The lysate was then incubated with 0.5 mlNi⁺²-NTA (Qiagen) resin as directed by the manufacturer. The protein waseluted with 20 mM sodium phosphate (pH 6) containing 500 mM NaCl and 200mM imidazole, and stored at −80° C. TABLE 1 Baculovirus ExpressionVectors Virus Tag Base Vector Cak1 None pVL Cdc4 None pBBIII Cdc4ΔWDNone pBBIII Cdc4^(F) C-terminal Flag pBBIII Cdc34 None pBBIII Cdc53^(M)N-terminal Myc pBBIII Clb5 None pVL Cln1^(HA) C-terminal HA pBBIIICln2^(HA) C-terminal HA pVL Gst-Cdc28^(HA) N-terminal Gst pVL C-terminalHA Gst-Cdc28^(HA) (D154N) N-terminal Gst pVL C-terminal HA Grr1^(G10)N-terminal His₆-G10 pBBHis His⁶-Cks1 N-terminal His₆ pVL Sic1 NonepBBIII Skp1 None pVL Skp1^(F) N-terminal Flag pBBIII Gst-Skp1 N-terminalGst pVL

[0275] For recombinant protein expression and assembly of complexes,4×10⁵ insect cells (Hi5, Invitrogen) were infected with the indicatedvirus combinations for 40 hours. These combinations includedbaculoviruses expressing Myc-tagged Cdc53 (Cdc53^(M)), Cdc34, Cdc4, andSkp1. Cells were then harvested and disrupted in lysis buffer (50 mMTris-HCl (pH 7.5), 150 mM NaCl, 0.5% Nonidet P40, 10 mM NaF, 10 mMβ-glycerol phosphate, 1 mM PMSF, and 5 μg/ml each leupeptin, antipain,and aprotinin). For isolation of protein complexes, typically about 3 mlof lysis buffer was used per 0.5×10⁸ cells.

[0276] To examine the assembly of recombinant yeast proteins, 0.4 mllysate were typically derived from 2×10⁶ cells. In both cases, celllysates were centrifuged for 2 minutes at 14,000×g, prior to affinity-or immuno-purification. Immunopurification was performed by incubatingthe lysates at 4° C. for 2 hours with 4 μg of the anti-Myc or anti-G10antibody and 8 μl of Protein A-Sepharose, or with 8 μl of immobilizedanti-Flag antibodies (Kodak; See, Example 1). Immune complexes werewashed three times with 1 ml of lysis butter prior to SDS-PAGE.

[0277] For SDS-PAGE, an equal volume of 2× sample buffer (250 mM Tris(pH 6.8), 4% SDS, 20% glycerol, 10% 2-mercaptoethanol) was added to thesamples to be tested, and boiled for 2 minutes. Samples were thenelectrophoresed in 12% polyacrylamide gels with 35 mA constant current.Proteins were transferred to nitrocellulose filters using a BioRadtransfer apparatus in 50 mM Tris/glycine buffer (pH 8), containing 20%methanol, for three hours, at 350 mA. The nitrocellulose filters werethen blocked with 5% non-fat dry milk solution for 1 hour, followed byincubation overnight with primary antibody. The antibody dilution usedwas 1:1000 for anti-Cdc4, anti-Cdc34, anti-gene10, anti-Sic1, anti-myc,and anti-HA; the anti-Skp1 antibody was diluted 1:4000. Blots werewashed in TBST (20 mM Tris (pH 8), 100 mM NaCl, 0.5% Tween-20) for 30minutes, and then incubated with either goat anti-rabbit conjugatedhorseradish peroxidase (HRP) or rabbit anti-goat conjugated HRP(Promega), as appropriate, at a dilution of 1:25,000, for 30 minutes.Immunoblots were then washed with TBST for 30 minutes, and developedusing enhanced chemiluminescence detection (Amersham) as described bythe supplier.

[0278] As shown in FIG. 1A, in the presence of all four proteins(Cdc53^(M), Cdc34, Cdc4, and Skp1), anti-Cdc53^(M) complexes containedCdc4, Cdc34, and Skp1. However, in the absence of Skp1, only low levelsof Cdc4 bound to Cdc53^(M), regardless of the presence of Cdc34 (FIG.1A, lanes 7 and S). This result was confirmed through the analysis ofCdc53^(M) association with anti-Cdc4 immune complexes (See, FIG. 1B).Thus, Skp1 was shown to facilitate association of Cdc53 with Cdc4. Incontrast, both Skp1 and Cdc34 can simultaneously associate withCdc53^(M) in the absence of other yeast proteins (See; FIGS. 1A and 1C).Together, these data indicated that Cdc34, Cdc53, Skp1, and Cdc4 form amultiprotein complex.

[0279] A. Sic1/Clb5/Gst-Cdc28HA(K−) Complexes

[0280] Sic1/Clb5/Gst-Cdc28HA(K−) complexes were purified from 4×10⁸cells, as described by Connell-Crowley et al. (Connell-Crowley et al.,Mol. Biol. Cell., 8:287-301 [1997]). Briefly, eight T-150 flasks ofinsect cells (Highfive, Invitrogen) were infected with 1 ml each ofbaculoviruses expressing either GST-Cdc28HA, Cln1HA, Cks1, and Cak1, orbaculoviruses expressing Gst-Cdc28HA(K−), Clb5, and Sic1. After 40hours, the cells were lysed at 4° C., in 6 ml of NETN (20 mM Tris-HCl,pH 8, 100 mM NaCl, 1 mM EDTA, 0.5% NP-40, 5 mM NaF, 30 mMp-nitrophenylphosphate, 1 μg/ml each leupeptin and antipain, and 1 mMPMSF). Lysates were cleared by centrifugation at 14,000×g for 10minutes. Supernatants were rotated with 0.2 ml of GSH-Sepharose for 60minutes at 4° C., and the beads were washed three times with 2 ml of thelysis buffer, followed by two washes with 100 mM Tris (pH 8), 100 mMNaCl. Proteins were then eluted with 0.2 ml of 100 mM Tris (pH 8), 100mM NaCl, 40 mM glutathione (Sigma), and 10% glycerol. The proteins werethen stored at −80° C. until use.

[0281] B. Gst-Cdc28HA/ClnHA/Cks1 and Gst-Cdc28HA(K−)/ClnHA/Cks1Complexes

[0282] Gst-Cdc28HA/ClnHA/Cks1 (i.e., “ClnHA/Gst-Cdc28HA/Cks1” in thelegend for FIG. 2A) and kinase impaired Gst-Cdc28HA(K−)/Cln1HA/Cks1complexes were prepared as described above, as were cells co-infectedwith viruses expressing appropriate proteins, and CAK1 expressing virusgenerated from a cDNA generously provided by C. Mann (See, Thuret etal., Cell 86:565-576 [1996]). The presence of Cks1 and Cak1 resulted ina 5-fold increase in the yield of active Cln/Cdc28 kinase complexes, aspurified after insect cell co-infection (determined using histone HI asa substrate). FIG. 2A shows an SDS-PAGE analysis of purifiedCdc28HA/Cks1. In this Figure, the asterisk indicates the position ofendogenous GST protein.

[0283] C. Phosphorylated Sic1 Complexes

[0284] Phosphorylated Sic1 complexes were generated by incubating 2.5 μMSic1/Clb5/Gst-Cdc28HA(K−) with Gst-Cdc28HA/Cln1HA/Cks1 (50 nM) and 1 mMATP in kinase buffer (50 mM Tris HCl (pH 7.5), 50 mM NaCl, 10 mM MgCl₂)for 45 minutes at 25° C. Control unphosphorylated Sic1 complexes wereproduced in an identical fashion by omitting Cln1 kinase. Cln/Cdc28autophosphorylation was performed by incubating 200 nM Cln/Cdc28complexes with 1 mM ATP in kinase buffer at 25° C. for 1 hour. Togenerate phosphorylated Sic1 free of Cln/Cdc28 kinase, bacterial Sic1(0.5 μM) was incubated with 2 mM ATP and Cln2/Gst-Cdc28/Cks1 immobilizedon GST-Sepharose (Pharmacia) for 60 minutes at 37° C. Forty ng ofphosphorylated Sic1 were removed from the beads for use inubiquitination reactions, at a final concentration of 1 nM. For³²P-labeling of Sic1 and Cln1 proteins, kinase reactions were performedat 25° C. for 30 minutes, using 50 μM (γ-³²P ATP (0.3 nCi/pmol))followed by incubation with 1 mM unlabeled ATP for an additional 30minutes.

[0285]FIG. 2B shows the gel results of phosphorylation of Sic1 byCln1/Cdc28 complexes in vitro. The result for Sic1/Clb5/Gst-Cdc28HA (K−)incubated with ATP are shown in lane 1, while the result for Cln1/Cdc28and ATP is shown in lane 2. Lane 3 shows the reaction products obtainedwhen Cln1/Cdc28 complexes alone were incubated with γ-³²P ATP. In lanes4 and 5, the results from experiments in which smaller amounts of Sic1phosphorylation reactions with 50 nM of Sic1 were performed in thepresence of γ-³²P ATP.

[0286] D. Grr1 Complexes

[0287] The Grr1 complexes were prepared by infecting one T-150 flask ofinsect cells as described above, with baculoviruses expressing Grr1G10,Skp1, and Cdc53^(M), or variations thereof. Forty hours after infection,the cells were lysed in 3 ml of NETN, and the lysates cleared bycentrifugation at 14,000×g for 10 minutes. Ten percent of each lysatewas used for immunoprecipitation with 5 μg of anti-gene 10 antibodies(Novagen), and 8 μl of protein A-Sepharose (4° C., for 90 minutes). Theimmune complexes were washed three times with 1 ml NETN prior to use inbinding experiments or ubiquitination reactions.

[0288] The complexes were immunoprecipitated with either (A) a Myc tagon Cdc53 (Cdc53^(M)) using anti-Myc antibodies or (B) a Flag tag on Cdc4(Cdc4^(F)) as described in Example 3. Immune complexes wereimmunoblotted and probed with anti-Myc to detect Cdc53^(M), anti-Cdc4,anti-Cdc34, and anti-Skp1 as described in Example 3 (See, FIG. 1).

EXAMPLE 3 In Vitro Binding Assays

[0289] Binding reactions were performed at 4° C. for 1 hour, in 100-250ml mixtures containing appropriate immunopurified complexes prepared asdescribed in Example 2, and affinity purified Sic1 (20 nM) or Cln (2 nM)complexes. Associated proteins were then washed three times with 1 ml oflysis buffer prior to SDS-PAGE and immunoblotting, were performed asdescribed above.

[0290] In some experiments, ³² P-labeled Sic1 or Cln complexes wereemployed at similar concentrations, and detected by autoradiography andphosphoimager analysis. Based on protein staining with Coomassie Blue orsilver, the quantities of proteins in anti-Skp1^(F) immune complex fromSkp1^(F)/Cdc53^(M)/Cdc4 expression cells was estimated to be: Skp1^(F)(1 μg), Cdc53^(M) (200 ng), and Cdc4 (200 ng). Likewise, the levels ofproteins in the anti-Grr1G10 complex were: Grr1G10 (100 ng), Cdc53^(M)(40 ng), and Skp1 (20 ng).

[0291] In additional experiments, insect cells were co-infected withconstant quantities of baculovirus expressing Skp1^(F) and increasingquantities of baculoviruses expressing either Cdc4, or a C-terminaltruncated form of Cdc4 lacking the last three WD-40 repeats (i.e.,Cdc4ΔWD; lanes 12-17). Lysates were immunoprecipitated with anti-Flagantibodies to precipitate Skp1^(F) complexes. Binding reactions withphosphorylated Sic1 complexes and detection of bound protein wereperformed as described above.

[0292]FIG. 2C indicates that phosphorylation of Sic1 is required for itsassociation with Cdc34/Cdc53/Skp1/Cdc4 complexes. As shown in thisFigure, phosphorylated Sic1 efficiently associates with Cdc53/Skp1/Cdc4complexes, and this association is dependent upon the presence of Skp1(See, FIG. 2C, lanes 6 and 8). Typically, 10-20% of the inputphosphorylated Sic1 was bound at about 20 nM Sic1. In contrast, theextent of binding of unphosphorylated Sic1 (lane 7) was comparable tothat observed in control immune complexes generated from uninfectedcells (lane 3) and was <1% of the input Sic1. Consistent with theresults in FIG. 1, the level of Cdc4 found in immune complexes lackingSkp1 were >10-fold lower than that found in the presence of Skp1. Thesedata suggest that Cdc4 and/or Skp1 function as binding factors for Sic1and that association of Sic1 with this complex requires phosphorylationby Cln1/Cdc28.

[0293]FIG. 2D shows that association of phosphorylated Sic1 with Cdc4 isenhanced by Skp1. In this Figure, lanes 3-9 contain anti-Flag immunecomplexes derived from cells infected with constant high quantities of abaculovirus expressing Cdc4^(F), while lanes 4-10 contain increasingquantities of a baculovirus expressing Skp1 in in vitro bindingreactions with purified Cln1/Cdc28-phosphorylated Sic1. While Skp1 alonedid not interact with Sic1, it stimulated association of Sic1 with Cdc4by about 5-fold (FIG. 2D). The weak association of Sic1 with Cdc4 alone(FIG. 2D, lane 3) may reflect the participation of an insect cell Skp1homolog. The results described herein clearly demonstrate a positivecontribution of Skp1 in the Cdc4/Sic1 interaction.

[0294]FIG. 2E shows that association of phosphorylated Sic1 with Skp1requires the WD-40 repeats of Cdc4. In this Figure, lanes 4-9 containproteins obtained from insect cells co-infected with constant quantitiesof baculovirus expressing Skp1^(F), and increasing quantities ofbaculoviruses expressing Cdc4, while lanes 12-17 contain lysates fromcells co-infected with constant quantities of baculovirus expressingSkp1^(F) and increasing quantities of baculoviruses expressing aC-terminal truncated form of Cdc4 lacking the last three WD-40 repeats(i.e., Cdc4ΔWD). Association of phosphorylated Sic1 with anti-Skp1^(F)immune complexes was absolutely dependent upon the presence of Cdc4(See, FIG. 2E, lanes 3 and 9). Moreover, deleting the last three WD-40repeats from the C-terminus of Cdc4 abolished its ability to associatewith phosphorylated Sic1 (FIG. 2E, lanes 10-16). Therefore, Cdc4functions as the specificity factor for binding of phosphorylated Sic1and the Cdc4-Sic1 interaction requires an intact WD-40 repeat domain inCdc4.

EXAMPLE 4 Ubiquitination Assays

[0295] In this Example, ubiquitination reactions were conducted. Inthese experiments, Ni²⁺-NTA resin was used to isolate ubiquitinatedproteins from extracts of wild-type cells or sic1 deletion mutantsexpressing His₆-Ub^(RA) or Ub^(RA) (Willems et al., [1996], supra). Inaddition, once the strategy to generate Cdc4/Skp1/Cdc53 complexes thatrecognized phosphorylated Sic1 was developed, experiments to determinewhether these complexes can catalyze ubiquitination of Sic1 in vitrowhen supplemented with Cdc34, E1, ATP, and ubiquitin were conducted.

[0296] In some experiments, bacterial Sic1 was used and where indicated,was phosphorylated with soluble or immobilized Gst-Cdc28HA/Cln2HA priorto use. Bacterial Sic1 ubiquitination reactions employed 100 nM yeast E1(a gift from S. Sadis and D. Firley, Department of Cell Biology, HarvardMedical School).

[0297] A. Ubiquitination of Sic1 In Vivo

[0298] To identify Sic1-ubiquitin conjugates in vivo, 200 ml (10⁷cells/ml) of wild-type (MT235), or a sic1 deletion (MT767) cellsexpressing either pCUP1-UB1^(RA) (<pUB204>) or pCUP1-UB1^(HIS-MYC-RA)(<pUB223>) were prepared, and lysates were generated in 50 mM Tris-HCl(pH 7.5), 100 mM NaCl, 0.1% NP-40, 1 mM PMSF, 0.6 mMdimethylaminopurine, 1 μg/ml leupeptin, 1 μg/ml pepstatin, 10 μg/mlTosylphenyl chloromethyl ketone and 10 μg/ml soybean trypsin inhibitoras described by Willems et al. (Willems et al., Cell 86:453-463 [1996]).Briefly, 8 μg of yeast protein was incubated with 12 μl of N+²NTA beads(Qiagen) for 1 hour at 4° C., as described by the manufacturer. Thebeads were then washed 3 times in lysis buffer, 1 time in high saltbuffer (50 mM Tris-HCl, pH 8.0, 0.5 M NaCl), and the proteins wereeluted with 10 μl of 100 mM Tris-HCl, pH 6.8, 1% SDS, 100 μM DTT, 100 μMEDTA. Proteins were separated by SDS-PAGE and immunoblotted withanti-Sic1 antibodies, as described above.

[0299]FIG. 3 shows that phosphorylated Sic1 is ubiquitinated in vivo andin vitro with purified Cdc34 E2 and Cdc53/Skp1/Cdc4 complexes. In thisFigure, the position of Sic1 and Sic1-ubiquitin conjugates are indicated(i.e., “Sic1” and “Sic1-Ub,” respectively).

[0300] B. Reconstitution of the Sic1 Ubiquitination Pathway UsingRecombinant Proteins

[0301] Ubiquitination reactions contained immune complexes prepared from2×10⁶ cells and equilibrated with ubiquitination buffer (100 mM Tris-HCl(pH 7.5), 5 mM MgCl₂, 0.6 mM DTT), 500 nM bacterial Cdc34, 300 nM humanE1 (a gift from M. Rolfe, Mitotix), 2 mM ATP, and 7 mM yeast ubiquitin(Sigma) or Gst-UB^(RA) (purified from bacteria expressing GEX-U6^(RA)[provided by M. Tyers, University of Toronto], and the method describedin Example 1 for GST-Skp1), and 80 ng of Sic1 complexes in a finalvolume of 14 μl, excluding bead volume. The human E1 purification wasdescribed by Rolfe et al. (Rolfe et al., Proc. Natl. Acad. Sci. USA92:3264-3268 [1995]). Reactions were allowed to proceed at 25° C. for 1hour or as indicated, quenched with 2× sample buffer (250 mM Tris (pH6.8), 4% SDS, 20% glycerol, and 10% 2-mercaptoethanol), and analyzed bySDS-PAGE and immunoblotting with anti-Sic1 antibodies as describedabove.

[0302] The results indicated that in the presence of all reactioncomponents, phosphorylated Sic1 was efficiently converted to highermolecular weight conjugates detectable with anti-Sic1 antibodies (See,FIG. 3B, lane 6; and FIG. 3C, lane 5). In contrast, unphosphorylatedSic1 was not detectably ubiquitinated. Sic1 ubiquitination absolutelyrequired Cdc34, Cdc4, Cdc53, Skp1, E1 and ubiquitin (See e.g, FIG. 3Band FIG. 3C). The pattern of high molecular weight Sic1 conjugatesobtained in reactions with ubiquitin was different from that observedwhen Gst-Ub^(RA) was used as the ubiquitination source as shown in lanes5 and 11 of FIG. 3C. These results confirm that the high molecularweight forms observed are products of ubiquitination. With Gst-Ub^(RA),the Sic1 reaction products were integrated into a ladder of bandsdiffering by approximately 35 kDa, the size of Gst-Ub^(RA) (See, FIG.3C, lane 11).

[0303] The ubiquitination reaction was time dependent and the reactionefficiency ranged from 10-40% of the input Sic1 protein (FIGS. 3B and3C). When the reaction was performed with pre-bound Sic1, the efficiencywas greater than 50%. In addition, greater than 50% of the Sic1ubiquitin conjugates formed after 60 minutes were found to havedissociated from the Cdc4/Skp1/Cdc53 complex. In addition, neitherGst-Cdc28, Clb5, Cdc53, Skp1, or Cdc4 formed ubiquitin conjugates underthe reaction conditions employed, although Cdc34 was ubiquitinated aspreviously reported (Haas et al., J. Biol. Chem., 266:5104-5112 [1991]).

[0304] C. Ubiquitination of Sic1 in Association with Clb5-Cdc28Complexes

[0305] To test whether Sic1 ubiquitination requires association withClb5/Cdc28 complexes, ubiquitination reactions using Sic1 produced inbacteria, with or without phosphorylation with Cln2/Cdc28 were performedas described above, with yeast E2 replacing human E1. To verify theabsence of Cln2HA/Cdc28HA in the ubiqutination reaction, Sic1 proteinswere also immunoblotted with anti-HA antibodies.

[0306] The results shown in FIG. 3D indicate that ubiquitination of Sic1does not require that Cln/Cdc28 be present in the ubiquitinationreaction, nor that Sic1 be associated with Clb5/Cdc28. In this Figure,lane 1 contains Sic1 purified from bacteria, while lane 2 contains Sic1treated with soluble Cln2/Gst-Cdc28, and lane 3 contains immobilizedCln2/Gst-Cdc28. Use of phosphorylated Sic1 that was free of Cln2 kinaseis indicated by an asterisk (lanes 3 and 9). As in the case of Sic1assembled in insect cells with Clb5/Cdc28, phosphorylated Sic1 frombacteria was efficiently ubiquitinated, with greater than 90% of theSic1 forming ubiquitin conjugates (lane 8), and ubiquitinationabsolutely required Sic1 phosphorylation (lane 4).

[0307] Although phosphorylation of Sic1 was required for its recognitionby Cdc4 and Skp1, it remained possible that Cln/Cdc28, present in smallamounts in the ubiquitination reaction, is also required for additionalsteps in the ubiquitination process, for instance, to phosphorylate theubiquitination machinery. To rule out this caveat, bacterial Sic1 wastreated with Cln2/Gst-Cdc28 complexes immobilized on Gst-Sepharosebeads, removed from the beads prior to use in ubiquitination reactions,and determined to be free of soluble kinase by immunoblotting withanti-HA antibodies. These results are shown in FIG. 3D, lane 3. Sic1phosphorylated in this manner was also efficiently ubiquitinated (See,FIG. 3D, lane 9). These data indicate that Sic1 phosphorylationconstitutes the primary requirement of Cln/Cdc28 kinases in Sic1ubiquitination in the in vitro reaction.

[0308] D. Clb5/Cdc28-phosphorylated Sic1 as a Substrate forUbiquitination

[0309] In these experiments, it was found that Clb5/Cdc28-phosphorylatedSic1 was also a substrate for ubiquitination. In these experiments,constant amounts of Sic1 were treated with increasing amounts ofClb5/Cdc28, until the kinase was in excess as determined by histonekinase assays. Under these conditions, Sic1 electrophoretic mobility wasreduced (FIG. 3E, lanes 1-6, top), as determined by immunoblotting.

[0310] Aliquots of differentially phosphorylated Sic1 were used inubiquitination reactions with immunopurified Cdc53^(M)/Cdc4/Skp1complexes supplemented with Cdc34, E1, ubiquitin, and ATP for 30minutes, as described above (See, FIG. 3E, lanes 1-6). As a negativecontrol, partially phosphorylated Sic1 corresponding to the Sic1 proteinin lane 5 (top) of FIG. 3E, was reacted in the absence of Cdc34 (lane 7)or the Cdc53^(M)/Cdc4/Skp1 complex (FIG. 3E, lane 8). Sic1ubiquitination was determined by immunoblotting with anti-Sic1antibodies (FIG. 3E, bottom).

[0311] Although Sic1 is an inhibitor of Cdc28/Clb5 complexes, when thekinase complex was in excess of Sic1, Sic1 was phosphorylated asdetermined both by reduced electrophoretic mobility (See, FIG. 3E) and³²P incorporation. This result may explain the fact that overexpressionof CLB5 can drive S-phase entry in cln- cells, and suggests that activeClb5/Cdc28 formed during Sic1 destruction may collaborate with Cln/Cdc28to complete the Sic1 ubiquitination process.

[0312] E. Sic1 Binding and Ubiquitination with Grr1

[0313] In these experiments, the Cdc4 was substituted with another F-boxprotein (Grr1) in order to determine if this protein could support Sic1binding and ubiquitination. Grr1 has an F-box near its N-terminus andcan interact simultaneously with Skp1 and Cdc53 when co-expressed ininsect cells (See e.g., Figure A).

[0314] These experiments were conducted as described above, with theexception being that Grr1 was substituted for Cdc4 (approximately 100ng). Proteins were separated by SDS-PAGE, and blotted with anti-Skp1anti-Myc to detect Grr1G10 and Cdc53^(M), with anti-Skp1 antibodies.

[0315] It was found that that Grr1 and Cdc4 with Skp1/Cdc53 are mutuallyexclusive. In contrast with Cdc4, it was not possible to demonstrateenhancement of the Grr1/Cdc53 interaction in insect cells byco-expression of Skp1, even though Skp1 assembled with these complexes.Importantly, Grr1 assembled with Cdc53/Skp1 complexes was unable toassociate with phosphorylated Sic1, and was unable to supportubiquitination of phosphorylated Sic1 complexes in the in vitro systemwith purified proteins under conditions where Cdc4 readily facilitatedSic1 binding and ubiquitination (See, FIGS. 4B and 4C). Therefore, F-boxproteins display selectivity toward particular targets.

[0316]FIG. 4A shows that Grr1 can associate with Skp1 and Cdc53, whileFIG. 4B shows that phosphorylated Sic1 associates with Cdc4 but notGir1-containing complexes. In this Figure, lanes 2-5 containanti-Skp1^(F) immune complexes derived from insect cells infected withthe indicated baculovirus combinations were used for binding reactionswith ³²P-labeled Sic1 complexes. Ten percent of the input Sic1 complex(lane 1) was included as a control. The presence of Cdc4, Skp1, Cdc53,and Grr1 was verified by immunoblotting.

[0317]FIG. 4C shows that Cdc4, but not Grr1, supports ubiquitination ofSic1 in vitro. The indicated anti-Skp1^(F) immune complexes were used inubiquitination assays as described for FIG. 3 (above) employingGst-Ub^(RA) as the ubiquitin source. Finally, FIG. 4 shows the resultsverifying the presence of reaction components derived fromimmunoprecipitation (in this Figure, the blot used for ubiquitinationassays was reprobed to detect Grr1G10, Cdc53^(M), and Cdc4).

EXAMPLE 4 Binding of Grr1 to Cln1 and Cln2

[0318] In this Example, the binding of Grr1 to Cln1 and Cln2 wasinvestigated. In particular, experiments were conducted in order todetermine whether Grr1 binds to Cln1 and/or Cln2 in aphosphorylation-dependent manner. Indeed, the finding that Sic1 isrecognized by the F-box protein Cdc4, together with a geneticrequirement for the F-box protein Grr1 in Cln destruction, led to theseexperiments to examine whether Grr1 functions in recognition ofphosphorylated Clns.

[0319] To generate Cln proteins for binding reactions, Cln/Gst-Cdc28/Ckscomplexes were isolated from insect cells as described in Example 2B. Inthe presence of ATP, both Cln1 and Cln2 are autophosphorylated, amodification that reduces their electrophoretic mobility (see below). Toexamine whether Grr1 can associate with phosphorylated Clns and tocompare the extent of selectivity of Grr1 and Cdc4 toward Cln binding,anti-Skp1^(F) immune complexes from cells co-expressing Grr1 or Cdc4 inthe presence or absence of Cdc53 prepared as described above, were usedin binding reactions with ³²P-labeled Cln1 or Cln2 kinase complexes.³²P-labeled Sic1 was used as a control for Cdc4 binding.

[0320] As shown in FIG. 5A, both Cln1 and Cln2 complexes associated withGrr1/Skp1^(F)/Cdc53 complexes with an efficiency of about 40% of theinput Cln1 or Cln2 (FIG. 5A, lanes 5 and 12), and this association didnot require Cdc53 (FIG. 5A, lane 16). In contrast, about 6% of the inputCln proteins associated with Cdc4/Skp1^(F) complexes independent of thepresence of Cdc53 (FIG. 5A, lanes 7, 11, and 15), compared with 1%association in the absence of an F-box protein (FIG. 5A, lanes 6, 10,and 14). The extent of selectivity of these F-box proteins for Cln andSic1 is further reflected by the observation that Cln1 protein presentin the phosphorylated Sic1 preparation was selectively enriched in Grr1complexes (FIG. 5A, lane 4). In this Figure, controls for the extent ofbinding (indicated by the asterisk) were 20% of input Cln and 10% ofinput Sic1. The presence of all proteins in the binding reaction wasconfirmed by immunoblotting (FIG. 5B; in this Figure, complexes used forbinding experiments in FIG. 5A were immunoblotted with the indicatedantibodies to verify the presence of Cdc4, Grr1G10, Cdc53^(M), andSkp1^(F)), and the quantities of Cdc4 and Grr1 were found to becomparable, based on Coomassie staining of SDS gels of immune complexes.Thus, Grr1 and Cdc4 display specificity toward physiological substrates.

[0321] Next, Grr1 alone or in complexes with Skp1 or Skp1/Cdc53 wereinmmunoprecipitated from insect cell lysates and used in binding assayswith phosphorylated or unphosphorylated Cln1 complexes prepared asdescribed above. The results are shown in FIG. 5C. As shown,unphosphorylated Cln1 was produced in insect cells as a complex withkinase deficient Gst-Cdc28(K−), which minimized Cln1 autophosphorylationduring expression, and allowed the role of phosphorylation to be tested.FIG. 5C, lane 1 shows that, as isolated, this Cln1 protein migrates as ahomogeneous species at approximately 66 kDa. In contrast, phosphorylatedCln1 (FIG. 5C, lane 2) undergoes a dramatic mobility shift toapproximately 80 kDa, consistent with in vivo observations. Lanes 4-11contain anti-Grr1G10 complexes derived from the indicated insect cellinfections used in binding reactions with either unphosphorylated Cln1HAcomplexes generated using kinase impaired Gst-Cdc28(K−)HA (FIG. 5C,lane 1) or phosphorylated Cln1HA/Gst-Cdc28HA complexes (FIG. 5C, lane2). Anti-HA antibodies were used to detect Cln1HA and Gst-Cdc28HA.Twenty percent of the input Cln1HA complexes were run as controls (FIG.5C, lanes 1 and 2). Cln1HA isolated from insect cells in complexes withactive Cdc28 migrated as a series of modified forms, reflecting partialphosphorylation of Cln in vivo in insect cells (See, FIG. 2A).Incubation of such ClnHA/Cdc28HA complexes with ATP quantitativelyshifts Cln1 HA to a single form migrating as an approximately 84 kDaprotein. The blot was reprobed to verify the presence of Grr1G10,Cdc53^(M), and Skp1^(F).

[0322] Phosphorylated Cln1 (and its associated Cdc28 protein)efficiently associated with all Grr1 complexes (FIG. 5C, lanes 6, 8, and10), but was absent from control binding reactions lacking Grr1 (FIG.5C, lane 4). In contrast, the levels of unphosphorylated Cln1 associatedwith Grr1 complexes were comparable to that found in binding reactionslacking Grr1 (FIG. 5C, lanes 3, 5, 7, and 9).

[0323] It was also determined that purified Skp1/Cdc53/Grr1 complexesare not sufficient for Cln1 ubiquitination by Cdc34 in vitro. Thus,association of both Cln1 with Grr1 and Sic1 with Cdc4 was found to begreatly enhanced by phosphorylation. Anti-Skp1^(F) immune complexes werepurified from insect cells infected with the indicated baculoviruses andsupplemented with E1, Cdc34, Gst-Ub^(RA), ATP, and either ³²P-labeledSic1 or Cln1, as described above (e.g., FIG. 3).

[0324] As shown in FIG. 5D, although the Grr1/Skp1/Cdc53 complex iscapable of binding efficiently to phosphorylated Cln1, it was notcompetent for Cln1 ubiquitination when supplemented with Cdc34 and E1.Moreover, FIG. 5D shows that Cdc4 complexes that functioned in Sic1ubiquitination also failed to catalyze ubiquitination of Cln1, despitethe fact that Cln1 can associate, albeit weakly, with Cdc4 (See, FIG.5A). In contrast, identical preparations of phosphorylated Cln1 proteinwere efficiently ubiquitinated in partially purified yeast lysates in aCdc34 dependent manner (FIG. 5E), indicating that this preparation ofCln1 is competent for ubiquitination.

EXAMPLE 5 Ubiquitination of Phosphorylated Cln1

[0325] In this Example, preparations of phosphorylated Cln1 (asdescribed above), were ubiquitinated in partially purified yeast lysatesin a Cdc34-dependent manner.

[0326] In these experiments, 0-100 μg YFII (a 250 mM NaCl eluate from aDEAE-cellulose column prepared exactly as described in Deshaies et al.[1995], supra) was supplemented with 500 nM Cdc34, 100 nM human E1,ubiquitin, and an ATP regenerating system (2 mM ATP, 600 mM creatinephosphate, and 0.15 mg/ml creatine kinase). The ubiquitination reactionwas initiated by addition of 20 ng Cln1HA/Gst-Cdc28HA/Cks1. Afterincubation for 60 minutes at 25° C., the reactions were quenched andimmunoblotted with anti-HA antibodies to detect Cln1HA and Gst-Cdc28HA.In FIG. 5E, the protein indicated by an asterisk is a yeast protein inYFII that cross-reacts with the anti-HA antibodies used. As indicated inthis Figure, this preparation of Cln1 is competent for ubiquitination.

EXAMPLE 6 Identification of Human F-Box Proteins

[0327] In this Example, new human F-box proteins were identified, usinga two hybrid system. The SKPI open reading frame (as an NdeI/BamHIrestriction fragment) was subcloned into pAS2 (See, Harper et al., Cell75:805-816 [1993]). pAS2-SKP1 was transformed into yeast strain Y190,and this strain was then used in a two hybrid screen with a human breastcDNA library generated in λACTII as described by below.

[0328] Yeast strain Y190 was deposited with the ATCC and assigned number(96400). Y190 was grown in YPD medium (10 g/l yeast extract, 20 g/lpeptone and 20 g/l dextrose) containing 10 mg/ml cycloheximide or on YPDplates (YPD medium containing 20 g/l agar) containing 10 mg/mlcycloheximide. Y190 contains two chromosomally located reporter geneswhose expression is regulated by Gal4.

[0329] The first reporter gene is the E. coli lacZ gene which is underthe control of the GAL1 promoter. The second reporter gene is theselectable HIS3 gene which encodes the enzyme imidazole glycerolphosphate (IGP) dehydrogenase. Yeast cells which express the HIS3 geneproduct can be selected by their ability to grow in medium lackinghistidine (i.e., SC-his medium). The λ ACTII phage cloning vector wasdeposited with the ATCC and assigned number 87006. This λ ACTII phagecloning vector was deposited as a lysogen in JM107 cells which are grownin LB containing 50 μg/ml ampicillin.

[0330] Yeast cells (strain Y190) containing specific nutritional markerswere grown on SC medium lacking one or more amino acids. SC mediumlacking a particular amino acid is referred to as dropout media. SCmedium is made using the following components: 10×YNB (67 g yeastnitrogen base without amino acids in 1 liter water, filter-sterilizedand stored in the dark).

[0331] Dropout mixture components: adenine  800 mg arginine  800 mgaspartic acid 4000 mg histidine  800 mg leucine 2400 mg lysine 1200 mgmethionine  800 mg phenylalanine 2000 mg threonine 8000 mg tryptophan 800 mg tyrosine 1200 mg uracil  800 mg

[0332] To make a dropout mixture, the above components are weighed out,leaving out the amino acids to be selected for, combined, and groundinto a fine powder using a mortar and pestle.

[0333] SC-Trp plates comprise per liter: 870 mg dropout mixture (minustryptophan), 20 g dextrose, 1 ml 1N NaOH, 20 g agar, water to 900 ml.The mixture is then autoclaved. After autoclaving, 100 ml 10×YNB isadded just prior to pouring the plates.

[0334] The bacterial strain used was E. coli strain BNN132 (ATCC 47059).These cells were grown in LB (10 g/l bacto-tryptone [DIFCO], 5 g/lbacto-yeast extract [DIFCO], 10 g/l NaCl, pH adjusted to 7.0 with NaOH).E. coli strain BL21(DE3) (Invitrogen) was grown in LB.

[0335] As described in more detail below, the pAS2/Skp1/Y190 strain wastransformed with 0.05 mg of plasmid library and 5 mg of carrier totalyeast RNA, and transformants were plated on a minimal media lackinghistidine, leucine, and tryptophan, but containing 25 mM3-aminotriazole. After 5 days at 30° C., plasmids were recovered fromβ-galactosidase positive colonies (See, Harper et at, Cell 75:805-816[1993]).

[0336] Also as described in more detail below, sequencing of cDNAinserts from positive plasmids revealed the presence of one cDNAcontaining significant sequence identity to Cdc4 in the F-box domain ofCdc4. This cDNA is referred to as “F3 gamma.” Other F-box containing,cDNAs were identified by searching the EST (expressed sequence tag)database, with the F-box region from F3 gamma. As novel F-box containingproteins were identified these were used to further search the ESTdatabase, in order to identify other novel F-box proteins. For some ofthese, both the human and mouse homologs were identified. It iscontemplated that these new F-box proteins act as components of E3complexes in mammalian cells (i.e., analogous to Cdc4 in budding yeast).Table 2 below lists the protein sequences identified in theseexperiments, while Table 3 provides the corresponding DNA sequences.FIG. 7 provides the alignments of these F-box proteins, with gapsindicated by dashes. Table 4 provides longer (i.e., more complete) cDNAand amino acid sequences for some of the F-box proteins identified inthe preliminary experiments. The sequences included in Table 4 containat least a large portion of the open reading frames (ORFs), and containpotential target binding domains. Both F1 and F2 contain leucine richrepeats (e.g., similar to Grr1). Thus, the present invention providesnumerous sequences suitable for detection and identification ofadditional F-box proteins, as well as targets for intervention in theproteolysis pathways (e.g., for drugs and other compounds suitable foruse to either enhance or reduce the efficiency and/or function of theF-box).

[0337] A. Generation of Human Breast Tissue cDNA Library in pACTII

[0338] In order to facilitate the isolation of F-box gene sequencesusing the yeast two-hybrid system, an human breast tissue cDNAexpression library was constructed in the λ ACTII phage cloning vector.This cloning vector allows for the construction of cDNA libraries fusedto sequences encoding the Gal4 transcriptional activation domain. Thephage can be converted to a plasmid form (pACTII) as described below.

[0339] An human breast tissue cDNA library was constructed using λ ACTIIas follows. Total RNA from breast tissue of an adult female obtainedfrom reductive mammoplasty was provided by Dr. Anne Bowcock (Universityof Texas Southwestern Medical Center). PolyA+ mRNA was produced using anmRNA isolation system (GIBCO-BRL). cDNA synthesis was accomplished usinga directional cDNA synthesis kit from Stratagene as described by themanufacturer.

[0340] After the synthesis of the second strand, the cDNA (in a volumeof 400 μl) was spermine precipitated by the addition of 22 μl of 100 mMspermine. The mixture was incubated on ice for 30 min and then pelletedby centrifugation in a microcentrifuge (Eppendorf) for 15 min at 4° C.The cDNA pellet was washed three times for 30 min/wash with 1 ml ofspermine wash buffer (70% ethanol, 10 mM Mg (Ac)₂, 0.3 M NaAc at pH 7)and once with 1 ml of 70% ethanol. The cDNA was then dissolved in 50 μlof TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA).

[0341] The ends of the cDNA were made blunt by treatment with T4 DNApolymerase using conditions recommended by the manufacturer(Stratagene). Following treatment with T4 DNA polymerase, 5 μl of 0.5 MEDTA was added and the mixture was extracted with phenol/chloroform andprecipitated with ethanol.

[0342] The precipitated cDNA, approximately 4 μg, was resuspended in 7μl of TE buffer and then ligated to 2 μg of a kinased adapteroligonucleotide in a total volume of 10 μl at 4° C. overnight (12-18hr). The hybridized oligonucleotide pair contained an EcoRI overhang.For example, the oligonucleotide CGCGCG hybridized with AATTCGCGCG (SEQID NO:59) will create a suitable EcoRI linker.

[0343] Following the ligation reaction, 170 μl of TE buffer, 20 μl of 1M KCl and 10 μl of 100 mM spermine were added. The mixture was incubatedon ice for 30 min and precipitated and washed as described above. Theadapted cDNA was resuspended in 20 μl of TE buffer and digested withXhoI prior to electrophoresis on a 1% LMA gel. cDNA having a length of600 bp or longer was excised from the gel and purified using standardtechniques prior to ligation into λ ACTII arms.

[0344] cDNA (0.1 μg) was ligated with 2 μg of λ ACTII plasmid DNAprepared as follows. One hundred micograms of λ ACTII plasmid DNA wasdigested with XhoI and EcoRI; the digestion products were thenprecipitated with ethanol, briefly dried and then resuspended in 190 μlof TE buffer. Ten microliters of 10 mM spermine were added to the sideof the tube and the contents were mixed by rapid inversion of the tube.An immediate and obvious precipitate formed and was pelleted bycentrifugation for 2 sec in a tabletop microcentrifuge. The pellet wasthen washed with spermine wash buffer followed by a wash with 70%ethanol as described above. The washed pellet was resuspended in 100 μlof TE buffer. This preparation of λ ACTII plasmid DNA was then used forligation into the cDNA containing adapters (prepared as describedabove).

[0345] The ligation of the adapted cDNA and digested λ ACTII plasmid DNAwas performed in a volume of 4 μl at 4° C. overnight. The ligationmixture was packaged using one Gigapack Gold packaging extract(Stratagene) according to the manufacturer's instructions. Approximately1×10⁸ total recombinants were obtained. The phage library was amplifiedon the LE392 strain of E. coli (Stratagene).

[0346] Automatic subcloning conversion of the cDNA library in λ ACTIIinto plasmid (pACTII) was accomplished by the incubation of 10⁹ phageparticles with 2 ml of a fresh overnight culture of E. coli strainBNN132 (ATCC 47059) in 10 mM MgCl₂ for 30 min at 30° C. without shaking.Two milliliters of LB (10 g/l bacto-tryptone, 5 g/l bacto-yeast extract,10 g/l NaCl, pH adjusted to 7.0 with NaOH) was then added and the cellswere incubated with shaking for 1 hr at 30° C. The cells were thenplated on ten 150 mm LB plates (15 g/l bacto-agar in LB) containing 50μg/ml ampicillin and incubated overnight at 37° C.

[0347] Ampicillin-resistant cells were collected by scraping the plates;the cells were then added to 3 liters of terrific broth (12 g/lbacto-tryptone, 24 g/l bacto yeast extract and 100 ml/1 of a solutioncomprising 0.17 M KH₂PO₄, 0.72 M K₂HPO₄) containing 50 μg/ml ampicillin.The culture was grown to stationary phase and plasmid DNA was isolatedusing CsCl density gradients by standard methods (J. Sambrook et al.,supra, at pp. 1.33-1.48)

[0348] B. Isolation of F-Box Sequences

[0349] In this portion of the Example, an improved version of the yeasttwo-hybrid system was employed to identify proteins that bind to Skp1.The two-hybrid system employs genetic selection to allow the isolationof interacting proteins. The use of genetic selection for the detectionof interacting proteins allows much larger cDNA libraries to be screenedfor associating clones than could be accomplished using other techniques(i.e., screening expression libraries, such as λgt11, with labelledproteins).

[0350] The improved two-hybrid system employs the yeast strain Y190 asthe recipient cell line. The yeast strain Y190 contains twochromosomally located reporter genes whose expression is regulated byGal4. The first reporter gene is the E. coli lacZ gene, which is underthe control of the GAL1 promoter. The second reporter gene is theselectable HIS3 gene. The two-hybrid system is improved by the use of anadditional assay to eliminate false positives. “False positives” aredefined as library clones that activate transcription in cellsexpressing fusions unrelated to the target protein (i.e., Skp1). Toisolate interacting proteins, Y190 cells are first transformed with afirst expression plasmid which encodes a fusion protein comprising ahybrid between the DNA-binding domain of the yeast transcription factorGal4 (amino acids 1-147) and a target protein (i.e., a protein which isused to identify proteins capable of interacting with this targetprotein). The transformed Y190 cells are next analyzed to determine theeffect of the introduction of the first expression plasmid. If thetransformation of Y190 cells with the expression plasmid which expressesthe target protein does not activate either the HIS3 or lacZ reportergenes, this transformed strain can now be used for screening anactivation domain cDNA library.

[0351] The activation domain library comprises plasmids capable ofexpressing the second hybrid molecules of the two-hybrid system. Thesecond hybrids comprise fusion proteins containing the sequencesencoding the Gal4 activation domiain II (amino acids 768-881) fused to acDNA library generated from human breast tissue (described above). Whenthe Y190 cells transformed with the first expression plasmid aretransformed with a second expression vector (from the expressionlibrary) capable of expressing a protein or portion of a protein whichcan bind to the Skp1 hybrid, transcription of the His3 and lacZ genes isactivated as the binding of the second hybrid brings the Gal4 activationdomain II in close proximity to the DNA binding domain of the Gal4protein which is bound to the UAS_(G) upstream of the His3 and lacZgenes on the chromosome.

[0352] In this two-hybrid system, Y190 cells were transformed with theexpression plasmid pAS2-Skp1 using lithium acetate according to standardtechniques (F. M. Ausubel, et al., Short Protocols in Molecular Biology,John Wiley & Sons, New York [1992], pp. 13-29-13,30). The expressionplasmid, pAS2-Skp1 encodes a fusion protein comprising a hybrid betweenthe DNA-binding domain of the yeast transcription factor Gal4 (aminoacids 1-147) and the Skp1 molecule. This first hybrid acts as “bait” forthe second hybrid molecule; the Gal4/Skp1 hybrid binds to the upstreamactivating sequence from GAL1 (UAS_(G)) sequences located upstream ofthe His3 and lacZ genes in the host cell chromosome. Because theGAL4-Skp1 hybrid lacks trans-activating sequences, Y190 cellstransformed with pAS1-Skp 1 were His⁻ and white.

[0353] Y190 cells were transformed with the pAS2-Skp1 plasmid asfollows. Y190 cells were grown in 5 ml of YPD medium (10 g/l yeastextract, 20 g/l peptone and 20 g/l dextrose) overnight to saturation at30° C. The next day, a liter sterile flask containing 300 ml of YPADmedium (YPD containing 30 mg/l adenine hemisulfate) was inoculated withthe overnight culture and grown overnight at 30° C. to a density of1×10⁷ cells/ml. The cells were then collected by centrifugation at4000×g for 5 min at room temperature. The cell pellet was then washed byresuspending the cells in 10 ml sterile H₂O followed by centrifugationat 5000×g for 5 min at room temperature. The washed cells wereresuspended in 1.5 ml of LiAc TE (1 vol 10×TE buffer [100 mM Tris-HCl,10 mM EDTA], pH 7.5), plus 1 vol of 10×LiAc stock solution (1M lithiumacetate, pH 7.5) plus 8 vol sterile H₂O. Five micrograms of pAS2-Skp1DNA and 200 μg carrier DNA (single-stranded, high molecular weightcarrier DNA was prepared from salmon sperm DNA using standard protocols;yeast total RNA may also be used as a carrier) were placed in a sterile1.5 ml microcentrifuge tube in a total volume of 20 μl. Two hundredmicroliters of the yeast suspension was added to the tube followed bythe addition of 1.2 ml of a LiAcPEG solution (8 vol of 50% (w/v)polyethylene glycol, MW 3350 plus 1 vol of 10×TE buffer, pH 7.5 plus 1vol 10×LiAc stock solution). The cells were then shaken for 30 min at30° C., followed by a heat shock (15 min at 42° C.). Following the heatshock, the cells were collected by centrifugation for 5 sec at roomtemperature in a tabletop microcentrifuge. The cell pellet was theresuspended in 1 ml of TE buffer and 200 μl of the suspension werespread onto SC-Trp medium.

[0354] The transformed Y190 cells (Y190/pAS2-Skp1) were then transformedwith a pACTII-human breast tissue cDNA library as described below. Theplasmids contained within this library encode the second hybrids of thetwo-hybrid system. The second hybrids comprised fusion proteinscontaining the sequences encoding the Gal4 activation domain II (aminoacids 768-881) fused to a cDNA library generated from human breasttissue. When a Y190/pAS2-Skp1 cell is transformed with a pACTIIexpression vector capable of expressing a protein or portion of aprotein which can bind to the Gal4-Skp1 hybrid, transcription of theHis3 and LacZ genes is activated as the binding of the second hybridbrings the Gal4 activation domain II in close proximity to the DNAbinding domain of the Gal4 protein which is bound to the UAS_(G)upstream of the His3 and lacZ genes on the chromosome.

[0355] Y190/pAS2-Skp1 cells were transformed with the pACTII-humanbreast cDNA library as follows. Briefly, the recipient strain(Y190/pAS2-Skp1 cells) were grown to mid-log phase (1×10⁷ cells/ml) inSC-Trp medium (SC medium lacking tryptophane). The OD₆₀₀ of this culturewas determined and 1 liter of YPD medium was inoculated with enough ofthe culture such that in 2 generations the cell density became 1×10⁷cells/ml. The cells where pelleted by centrifugation and the pellet wasresuspended in LiAcTE (the volume is not critical as this is a washstep). The cells were pelleted by centrifugation and the cells wereresuspended in 25 ml of LiSORB (100 mM LiAc, 10 mM Tris-HCl (pH 8.0), 1mM EDTA, 1 M sorbitol). The cells were then incubated for 30 min at 30°C. with shaking. The cells were then pelleted by centrifugation asdescribed above and resuspended in 2.5 ml of LiSORB. After removing 100μl of cells for a negative control, 50 μg of pACTII library DNA and 5 mgof yeast total RNA carrier was added. The mixture was mixed well andthen incubated for 10 min at 30° C. without shaking. The cells were thentransferred to a 250 ml flask and 22.5 ml of LiAcPEG (LiAcTE containing40% polyethylene glycol, MW 3350) was added and the suspension was wellmixed. The flask was then placed in a 42° C. water bath for 12 min toheat shock the cells. Following the heat shock, the transformationmixture was added to 500 ml of SC-Trp, -Leu, -His medium and the culturewas allowed to recover at 30° C. for 4 hours; at this point the cellsare established as transformants. Next, 4×10⁵ transformants wereobtained by transformation of 1×10¹⁰ Y190/pAS2-Skp1 cells with 50 μg ofpACTII library DNA.

[0356] Transformants were subjected to selection for histidineprototrophy by plating 300 μl of the culture on 15 cm petri dishescontaining SC-Trp, -Leu, -His medium containing 50 mM 3 amino-triazole(Sigma), and incubated for 30° C. for 3-5 days.

[0357] The rare surviving colonies were screened for their ability toproduce β-galactosidase using a filter lift assay (L. Breeden and K.Nasmyth, Cold Spring Harbor Symp. Quant. Biol., 50:643-650 [1985]).Briefly, colonies were transferred onto nitrocellulose filters(Scheicher and Schuell, BA85 45 μm circular filters) by laying thefilters onto plates containing the yeast colonies and allowing thefilter to wet completely. The filters were lifted off the plates andthen submerged in liquid nitrogen for 5-10 sec. The filters were thenplaced cell side up into a petri dish containing 3 MM chromatographypaper (Whatman) saturated 0.3 ml/square inch with a solution comprising100 mM sodium phosphate, pH 7.0, 10 mM KCl, 0.7 mM magnesium sulfate, 10mM 2-mercaptoethanol and 1 mg/ml X-gal(5-bromo-4-chloro-3-indolyl-β-galactoside). The filters were incubatedat 30° C. until blue color developed (30 min to overnight). Positive(i.e., blue) colonies were then patched onto a master plate for furtheranalysis.

[0358] Plasmids recovered from 20 His+ blue (i.e., lacZ expressing)colonies were sequenced from their 5′ ends using the chain terminationmethod in conjunction with the Sequenase® enzyme (U.S. Biochemicals);sequencing was performed according the manufacturer's instructions. Theamino acid sequence was deduced from the nucleotide sequences locatedwithin the 20 inserts and were compared to sequences listed in theGenBank. One of these 20 clones were found to be related to the F-boxprotein met30 from S. cerevisiae in the F-box domain. The F-box regionof Fgamma was used to search the EST database of Genbank, and identifiedtwo novel mammalian F-box-containing cDNAs (F13 Omicron and F14 Pi), inaddition to two F-box proteins from C. elegans (C02FS7 and YK18A11). TheGenbank accession numbers for these four cDNAs are AA422959, AA462249,R12719, and D35163).

[0359] These novel F-box sequences were used to search the EST sequenceslisted in Genbank. This search yielded F7 Theta (AA008567), F1 alpha(F12916), F4 Delta (AA167804), TRCP (AA478504), F6 Eta (AA027176), F15Rho (AA538102), F8 Iota (AA295683), MD6 (AA145853), and Skp2 homologs(U33761). These F-box protein sequences were then used in additional ESTdatabase searches and yielded sequences for F2 Beta (H58848), F5 Zeta(R17328), F9 Kappa (AA459120), F10 Lambda (AA501293), F11 Mu (AA069757),F12 Nu (AA000239), F16 Sigma (H49462), F17 Tau (AA381895), and F18 Phi(AA309734). cDNA clones in bacterial plasmids including pBluescript(Stratagene) were retrieved from the EST cDNA Image Consortium andsubjected to sequence analysis using dideoxy DNA sequencing (See e.g.,Sambrook et al., supra) to verify the F-box sequences. For three cDNAs(F1 alpha, F2 beta, and F4 delta, complete sequences of the availablecDNA clones were obtained by standard primer walking (See e.g., Sambrooket al., supra).

[0360] This approach finds use in identification of other novel F-boxcontaining proteins, either using cDNA libraries from other tissues orby performing additional database searches. The use of cDNA librariesfrom various tissues allows the identification of F-box proteins thatare cell-type specific. Additional alternative approaches, such asexpression screening of λgt11-based plaque libraries with Skp1 proteinare also contemplated as methods for yielding novel F-box proteins.TABLE 2 F-Box Sequences Identified Name, Source & Genbank # Amino AcidSequence SEQ ID NO: TRCP LPARGLDHIAENILSYLDAKSLCAAELVCKEWYRVTSDGMLW SEQID NO:1 (human) AA478504 F1 (Alpha)LPKELLLRIFSFLDIVTLCRCAQISKAWNILALDGSNW SEQ ID NO:3 (human) F12916 F2(Beta) LPYELIQLILNHLTLPDLCRLAQTCKLLSQHCCDPLQY SEQ ID NO:5 (human) H58848F2 (Beta) LPYELIQLILNHLSLPDLCRLAQTCRLLHQHCCDPLQY SEQ ID NO:7 (mouse) F3(Gamma) LPTDPLLLILSFLDYRDLINCCYVSRRLSQLSSHDPLW SEQ ID NO:9 (human)AA101399 F3 (Gamma) LPTDPLLLIVSFVDYRDLINCCYVSRSVSQLSTHDPLW SEQ ID NO:11(mounse) F4 (Delta) LPPEVMLSIFSYLNPQELCRCSQVSMKWSQLTKTGSLW SEQ ID NO:13(human) AA167804 F4 (Delta) LPPEVMLSIFSYLNPQELCRCSQVSTKWSQLAKTGSLW SEQID NO:15 (mouse) F5 (Zeta) LPLEMLTYILSFLPLSDQKEASLVSWAWYRAAQNALRERLW SEQID NO:17 (human) R17328 F6 (Eta) LPPELSFTILSYLNATDLCLASCVWQDLANDELLW SEQID NO:19 (human) AA027176 F6 (Eta) LPPELSFTILSYLNAIDLCLASCVWQDLANDELLWSEQ ID NO:21 (mouse) AA213046 F7 (Theta)LPRVLSVYIFSFLDPRSLCRCAQVSWYWKSLAELDQLW SEQ ID NO:23 (mouse) AA008567 F8(Iota) LPIDVQLYILSFLSPHDLCQLGSTNHYWNETVRHPILW SEQ ID NO:25 (human)AA295683 F9 (Kappa) LPLELWRMILAYLHLPDLGRCSLVCRAWYELILSLDSTRW SEQ IDNO:27 (human) AA459120 F10 (Lambda) LPAEITFKIFSQLDIRSLCRASLTCRSWNDFKSSEQ ID NO:29 (mouse) AA501293 F11 (Mu)LPLLQQPLLCSVAHPIASFTMLSYLTGKEAAHLSVELW SEQ ID NO:31 (mouse) AA069757 F12(Nu) LPDSLVYQIFLSLGPADVLAAGLVCRQWQAVSRDEFLW SEQ ID NO:33 (mouse)AA000239 F13 LPEEVLALIFRDLPLRDLAVATRVCRAWAAA SEQ ID NO:35 (Omicron)(mouse) AA422959 F14 (Pi) LPSVPMMEILSYLDAYSLLQAAQVNKNWNELASSDVLW SEQ IDNO:37 (mouse) AA462249 F15 (Rho) MPSEILVKILSYLDAVTLVCIGCVSRRFYHLADDNLIWSEQ ID NO:39 (mouse) AA538102 F16 (Sigma)LPMEVLMYIFRWVVSSDLDLRSLEQLSLVCRGFYICARDPEIW SEQ ID NO:41 (human) AA49462F16 (Sigma) LSLVCRGFYICARDPEIW SEQ ID NO:43 (mouse) AA410485 F17 (Tau)LPYELAINIFXYLDRKELGRCAQVSKTWEGD SEQ ID NO:45 (human) AA381895 F18 (Phi)LPLELKLRIFRLLDVRSVLSLSAVCRDLFTASNDPLLW SEQ ID NO:47 (mouse) AA309734 F18(Phi) LPLELKLRIFRLLDVHSVLALSAVCHDLLIASNDPLLW SEQ ID NO:49 (human) W20645MD6 LPLELSFYLLKWLDPQTLLTCCLVSKQWNKVISACTEVW SEQ ID NO:57 (human)

[0361] TABLE 3 F-Box Sequences Identified Name and SEQ ID Source DNASequence NO: TRCP CTGCCAGCTCGGGGATTGGATCATATTGC SEQ ID (human)TGAGAACATTCTGTCATACCTGGATGCCA NO:2 AATCACTATGTGCTGCTGAACTTGTGTGCAAGGAATGGTACCGAGTGACCTCTGATGG CATGCTGTGG F1 (Alpha)TTACCCAAAGAACTTCTGTTAAGAATATT SEQ ID (human)TTCCTTCTTGGATATAGTAACTTTGTGCC NO:4 GATGTGCACAGATTTCCAAGGCTTGGAACATCTTAGCCCTGGATGGAAGCAACTGG F2 (Beta) CTACCTTATGAGCTTATTCAGCTGATTCT SEQID (human) GAATCATCTTACACTACCAGACCTGTGTA NO:6 GATTAGCACAGAC F2 (Beta)CTACCATATGAGCTCATTCAACTGATTCT SEQ ID (mouse)GAATCATCTTTCACTACCAGACCTGTGTA NO:8 GATTAGCCCAGACTTGCAGGCTTCTCCACCAGCATTGCTGTGATCCTCTGCAATAT F3 (Gamma) CTGCCCACCGATCCCCTGCTCCTCATCTT SEQID (human) ATCCTTTTTGGACTATCGGGATCTAATCA NO:10ACTGTTGTTATGTCAGTCGAAGACTTAGC CAGCTATCAAGTCATGATCCGCTGTGG F3 (Gamma)CTACCCACCGACCCTCTGCTCCTCATAGT SEQ ID (mouse)ATCCTTCGTGGACTACAGGGACCTAATCA NO:12 ATTGTTGCTATGTTAGTCGAAGCGTTAGCCAGCTATCAACTCATGATCCACTGTGG F4 (Delta) CTTCCTCCTGAGGTAATGCTGTCAATTTT SEQID (human) CAGCTATCTTAATCCTCAAGAGTTATTCG NO:14ATGCAGTCAAGTAAGCATGAAATGGTCTC AGCTGACAAAAACGGGATCGCTTTGG F4 (Delta)CTTCCTCCTGAGGTAATGCTGTCCATTTT SEQ ID (mouse)CAGTTACCTTAATCCTCAAGAATTGTGTC NO:16 GGTGTAGTCAAGTCAGTACTAAGTGGTCTCAGCTGGCAAAAACAGGATCTTTGTGG F5 (Zeta) CTGCCCCTGGAGATGCTCACATATATTCT SEQID (human) GAGCTTCCTGCCTCTGTCAGATCAGAAAG NO:18AGGCCTCCCTCGTGAGTTGGGCTTGGTAC CGTGCTGCCCAGAATGCCCTTCGGGAGAG GCTGTGG F6(Eta) TTGCCTCCTGAGCTAAGCTTTACCATCTT SEQ ID (human)GTCCTACCTGAATGCAACTGACCTTTGCT NO:20 TGGCTTCATGTGTTTGGCAGGACCTTGCGAATGATGAACTTCTCTGG F6 (Eta) CTGCCTCCTGAGCTGAGCCTCACCATCCT SEQ ID (mouse)ATCCCACCTGGATGCAACTGACCTTTGCC NO:22 TAGCTTCCTGTGGTTGGCAAGAACTCGCTAATGATGAACTTCTCTGG F7 (Theta) CTTCCAAGGGTGTTATCTGTCTACATCTT SEQ ID(mouse) TTCCTTCCTGGATCCCCGGAGTCTTTGCC NO:24GTTGTGCACAGGTGAGCTGGTACTGGAAG AGCTTGGCTGAGTTGGACCAGCTCTGG F8 (Iota)CTGCCGATTGATGTACAGCTATATATTTT SEQ ID (human)GTCCTTTCTTTCACCTCATGATCTGTGTC NO:26 AGTTGGGAAGTACAAATCATTATTGGAATGAAACTGTAAGACATCCAATTCTTTGG F9 (Kappa) CTCCCCTTGGAGCTGTGGCGCATGATCTT SEQID (human) AGCCTACTTGCACCTTCCCGACCTGGGCC NO:28GCTGCAGCCTGGTATGCAGGGCCTGGTAT GAACTGATCCTCAGTCTCGACAGCACCCG CTGG F10(Lambda) CTGCCTGCAGAAATCACTTTTAAAATTTT SEQ ID (mouse)CAGTCAGCTGGACATTCGGAGTCTGTGCA NO:30 GGGCTTCATTGACATGCAGGAGCTGGAAT GACF11 (Mu) CTGCCATTACTGCAGCAGCCACTTCTGTG SEQ ID (mouse)TTCTGTGGCTCATCCCATCGCCAGCTTCA NO:32 CCATGCTGTCATACCTCACGGGAAAGGAGGCCGCTCATCTGTCAGTGGAGTTGTGG F12 (Nu) CTCCCCGACAGCCTTGTCTACCAGATCTT SEQID (mouse) CCTGAGTTTGGGCCCTGCAGATGTGCTGG NO:34CTGCTGGGCTGGTATGCCGCCAATGGCAG GCTGTGTCCCGGGATGAGTTCTTATGG F13CTGCCAGAGGAAGTGTTGGCGCTCATCTT SEQ ID (Omicron)CCGTGACCTGCCTCTCAGGGACCTTGCTG NO:36 (mouse)TAGCCACCAGAGTCTGCAGGGCCTGGGCG GCGGCT F14 (Pi)TTACCTAGTGTGCCGATGATGGAAATCCT SEQ ID (mouse)CTCCTATCTGGATGCCTACAGTTTGCTAC NO:38 AGGCTGCCCAAGTGAACAAGAACTGGAATGAACTTGCAAGCAGTGATGTCCTGTGG F15 (Rho) ATGCCATCGGAAATCTTGGTGAAGATACT SEQID (mouse) TTCTTACTTGGATGCGGTGACCTTGGTGT NO:40GCATTGGATGTGTGAGCAGACGCTTTTAT CATTTGGCTGATGACAATCTTATTTGG F16 (Sigma)CTGCCAATGGAGGTCCTGATGTACATCTT SEQ ID (human)CCGATGGGTGGTGTCTAGTGACTTGGACC NO:42 TCAGATCATTGGAGCAGTTGTCGCTGGTGTGCAGAGGGTTCTACATCTGTGCCAGAGA CCCTGAAATATGG F16 (Sigma)GACTTGGACCTCAGATCGTTAGAGCAGTT SEQ ID (mouse)GTCACTGGTGTGCAGAGGATTCTATATCT NO:44 GTGCCAGAGACCCTGAAATCTGG F17 (Tau)CTGCCTTACGAATTGGCAATCAATATATT SEQ ID (human)TNAGTATCTGGACAGGAAAGAACTAGGAA NO:46 GATGTGCACAGGTGAGCAAGACGTGGGAAGGTGATT F18 (Phi) CTCCCATTGGAACTGAAACTACGGATCTT SEQ ID (human)CCGACTTCTGGATGTTCGTTCCGTCTTGT NO:48 CTTTGTCTGCGGTTTGTCGTGACCTCTTTACTGCTTCAAATGACCCACTCCTGTGG F18 (Phi) CTTCCACTGGAGCTGAAACTACGCATCTT SEQID (mouse) CCGACTTTTGGATGTTCATTCTGTCCTGG NO:50CCCTGTCTGCAGTCTGTCATGACCTCCTC ATTGCGTCAAATGACCCACTGCTGTGG MD6CTTCCCCTGGAGCTCAGTTTTTATTTGTT SEQ ID (human)AAAATGGCTCGATCCTCAGACTTTACTCA NO:58 CATGCTGCCTCGTCTCTAAACAGTGGAATAAGGTGATAAGTGCCTGTACAGAGGTGTG G

[0362] TABLE 4 Sequences of Some F-Box Proteins Name & SEQ ID SourceSequence NO: F1 SAMVFSNNDEGLINKKLPKELLLRIFSFLDIVTLCR SEQ ID AlphaCAQISKAWNILALDGSNWQRIDLFNFQIDVEGRVVE NO:51 (human)NISKRCGGFLRKLSLRGCIGVGDSSLKTFAQNCRNIEHLNLNGCTKITDSTCYSLSRFCSKLKHLDLTSCVSITNSSLKGISEGCRNLEYLNLSWCDQITKDGIEALVRGCRGLKALLLRGCTQLEDEALKHIQNYCHELVSLNLQSCSRITDEGVVQICRGCHRLQALCLSGCSNLTDASLTALGLNCPRLQILEAARCSHLTDAGFTLLARNCHELEKMDLEECILITDSTLIQLSIHCPKLQALSLSHCELITDDGILHLSNSTCGHERLRVLELDNCLLITDVALEHLETAEAWSASSCTTASRLPVQASSGCGLSSLMSKSTPTLLPSPHRQQWQEVDSDCAGAVSFSDSSCLGP RGDEASFPLEDLSLPDRLHHHPIC F1TTCGGCCATGGTTTTCTCAAACAATGATGAAGGCCT SEQ ID AlphaTATTAACAAAAAGTTACCCAAAGAACTTCTGTTAAG NO:52 (human)AATATTTTCCTTCTTGGATATAGTAACTTTGTGCCGATGTGCACAGATTTCCAAGGCTTGGAACATCTTAGCCCTGGATGGAAGCAACTGGCAAAGAATAGATCTTTTTAACTTTCAAATAGATGTAGAGGGTCGAGTGGTGGAAAATATCTCGAAGCGATGCGGTGGATTCCTGAGGAAGCTCAGCTTGCGAGGCTGCATTGGTGTTGGGGATTCCTCCTTGAAGACCTTTGCACAGAACTGCCGAAACATTGAACATTTGAACCTCAATGGATGCACAAAAATCACTGACAGCACGTGTTATAGCCTTAGCAGATTCTGTTCCAAGCTGAAACATCTGGATCTGACCTCCTGTGTGTCTATTACAAACAGCTCCTTGAAGGGGATCAGTGAGGGCTGCCGAAACCTGGAGTACCTGAACCTCTCTTGGTGTGATCAGATCACGAAGGATGGCATCGAGGCACTGGTGCGAGGTTGTCGAGGCCTGAAAGCCCTGCTCCTGAGGGGCTGCACACAGTTAGAAGATGAAGCTCTGAAACACATTCAGAATTACTGCCATGAGCTTGTGAGCCTCAACTTGCAGTCCTGCTCACGTATCACGGATGAAGGTGTGGTGCAGATATGCAGGGGCTGTCACCGGCTACAGGCTCTCTGCCTTTCGGGTTGCAGCAACCTCACAGATGCCTCTCTTACAGCCCTGGGTTTGAACTGTCCGCGACTGCAAATTTTGGAGGCTGCCCGATGCTCCCATTTGACTGACGCAGGTTTTACACTTTTAGCTCGGAATTGCCACGAATTGGAGAAGATGGATCTTGAAGAATGCATCCTGATAACCGACAGCACACTCATCCAGCTCTCCATTCACTGTCCTAAACTGCAAGCCCTGAGCCTGTCCCACTGTGAACTCATCACAGATGATGGGATCCTGCACCTGAGCAACAGTACCTGTGGCCATGAGAGGCTGCGGGTACTGGAGTTGGACAACTGCCTCCTCATCACTGATGTGGCCCTGGAACACCTAGAAACTGCCGAGGCCTGGAGCGCCTCGAGCTGTACGACTGCCAGCAGGTTACCCGTGCAGGCATCAAGCGGATGCGGGCTCAGCTCCCTCATGTCAAAGTCCACGCCTACTTTGCTCCCGTCACCCCACCG ACAGCA F2RPRFGTSDIEDDAYAEKDGCGMDSLNKKFSSAVLGE SEQ ID BetaGPNNGYFDKLPYELIQLILNHLTLPDLCRLAQTCKL NO:53 (human)LSQHCCDPLQYIHLNLQPYWAKLDDTSLEFLQSRCTLVQWLNLSWTGNRGFISVAGFSRFLKVCGSELVRLELSCSHFLNETCLEVISEMCPNLQALNLSSCDKLPPQAFNHIAKLCSLKRLVLYRTKVEQTALLSILNFCSELQHLSLGSCVMIEDYDVIASMIGAKCKKLRTLDLWRCKNITENGIAELASGCPLLEELDLGWCPTLQSSTGCFTRLAHQLPNLQKLFLTANRSVCDTDIDELACNCTRLQQLDILGKVTIYKFVLNVCFLDRKANLRLFVRKKKI FGYNKNFILIRWLGLIGNAR F2AGGCCAAGATTCGGCACGAGTGATATAGAAGATGAT SEQ ID BetaGCCTATGCAGAAAAGGATGGTTGTGGAATGGACAGT NO:54 (human)CTTAACAAAAAGTTTAGCAGTGCTGTCCTCGGGGAAGGGCCAAATAATGGGTATTTTGATAAACTACCTTATGAGCTTATTCAGCTGATTCTGAATCATCTTACACTACCAGACCTGTGTAGATTAGCACAGACTTGCAAACTAMISTYCTGAGCCAGCATTGCTGTGATCCTCTGCAATACATCCACCTCAATCTGCAACCATACTGGGCAAAACTAGATGACACTTCTCTGGAATTTCTACAGTCTCGCTGCACTCTTGTCCAGTGGCTTAATTTATCTTGGACTGGCAATAGAGGCTTCATCTCTGTTGCAGGATTTAGCAGGTTTCTGAAGGTTTGTGGATCCGAATTAGTACGCCTTGAATTGTCTTGCAGCCACTTTCTTAATGAAACTTGCTTAGAAGTTATTTCTGAGATGTGTCCAAATCTACAGGCCTTAAATCTCTCCTCCTGTGATAAGCTACCACCTCAAGCTTTCAACCACATTGCCAAGTTATGCAGCCTTAAACGACTTGTTCTCTATCGAACAAAAGTAGAGCAAACAGCACTGCTCAGCATTTTGAACTTCTGTTCAGAGCTTCAGCACCTCAGTTTAGGCAGTTGTGTCATGATTGAAGACTATGATGTGATAGCTAGCATGATAGGAGCCAAGTGTAAAAAACTCCGGACCCTGGATCTGTGGAGATGTAAGAATATTACTGAGAATGGAATAGCAGAACTGGCTTCTGGGTGTCCACTACTGGAGGAGCTTGACCTTGGCTGGTGCCCAACTCTGCAGAGCAGCACCGGGTGCTTCACCAGACTGGCACACCAGCTCCCAAACTTGCAAAAACTCTTTCTTACAGCTAATAGATCTGTGTGTGACACAGACATTGATGAATTGGCATGTAATTGTACCAGGTTACAGCAGCTGGACATATTAGGTAAGGTTACAATATATAAATTTGTTTTAAATGTCTGTTTCCTTGACAGAAAAGCCAATCTCAGACTTTTTGTTAGGAAAAAGAAAATTTTTGGATACAATAAAAATTTTATCCTGATAAGATGGCTTGGTTTGATAGGAAATGCCAGATAGATCAGTTAATATAGGGAATAATTATATATGTACTTTAATAAAATAGTGAGGACAATAACAATTTTATAGTTGAACTGTAAAAAACTATAACCATTAATTCTTGGTCTACTTGTAAGAGTGAGAATTTACATGAGCTGCGCTCTCTATTTTTATTAAGGAGAGAAGAAATTAATTCATTTGTATAATGAATTCAAGCTAGTTTTTTTAAGTTTCTTAATTA AGCGGCCGCAAGCTTA F4WVIMLZERQKFFKYSVDEKSDKEAEVSEHSTGITHL SEQ ID DeltaPPEVMLSIFSYLNPQELCRCSQVSMKWSQLTKTGSL NO:55 (human)WKHLYPVHWARGDWYSGPATELDTEPDDEWVKNRKDESRAFHEWDEDADIDESEESAEESIAISIAQMEKRLLHGLIHNVLPYVGTSVKTLVLAYSSAVSSKMVRQILELCPNLEHLDLTQTDISDSAFDSWSWLGCCQSLRHLDLSGCEKITDVALEKISRALGILTSHQSGFLKTSTSKITSTAWKNKDITMQSTKQYACLHDLTNKGIGEEIDNEHPWTKPVSSENFTSPYVWMLDAEDLADIEDTVEWRHRNVESLCVMETASNFSCSTSGCFSKDIVGLRTSVCWQQHCASPAFAYCGHSFCCTGTALRTMSSLPESSAMCRKAARTRLPRGKDLIYFGSEKSDQETGRVLLFLSLSGCYQITDHGLRVLTLGGGLPYLEHLNLSGCLTITGAGLQDLVSACPSLNDEYFYYCDNINGPHADTASGC QNLQCGFRACCRSGE F4ATGGTAATCATGCTGTAAGAGCGACAGAAATTTTTT SEQ ID DeltaAAATATTCCGTGGATGAAAAGTCAGATAAAGAAGCA NO:56 (human)GAAGTGTCAGAACACTCCACAGGTATAACCCATCTTCCTCCTGAGGTAATGCTGTCAATTTTCAGCTATCTTAATCCTCAAGAGTTATGTCGATGCAGTCAAGTAAGCATGAAATGGTCTCAGCTGACAAAAACGGGATCGCTTTGGAAACATCTTTACCCTGTTCATTGGGCCAGAGGTGACTGGTATAGTGGTCCCGCAACTGAACTTGATACTGAACCTGATGATGAATGGGTGAAAAATAGGAAAGATGAAAGTCGTGCTTTTCATGAGTGGGATGAAGATGCTGACATTGATGAATCTGAAGAGTCTGCGGAGGAATCAATTGCTATCAGCATTGCACAAATGGAAAAACGTTTACTCCATGGCTTAATTCATAACGTTCTACCATATGTTGGTACTTCTGTAAAAACCTTAGTATTAGCATACAGCTCTGCAGTTTCCAGCAAAATGGTTAGGCAGATTTTAGAGCTTTGTCCTAACCTGGAGCATCTGGATCTTACCCAGACTGACATTTCAGATTCTGCATTTGACAGTTGGTCTTGGCTTGGTTGCTGCCAGAGTCTTCGGCATCTTGATCTGTCTGGTTGTGAGAAAATCACAGATGTGGCCCTAGAGAAGATTTCCAGAGCTCTTGGAATTCTGACATCTCATCAAAGTGGCTTTTTGAAAACATCTACAAGCAAAATTACTTCAACTGCGTGGAAAAATAAAGACATTACCATGCAGTCCACCAAGCAGTATGCCTGTTTGCACGATTTAACTAACAAGGGCATTGGAGAAGAAATAGATAATGAACACCCCTGGACTAAGCCTGTTTCTTCTGAGAATTTCACTTCTCCTTATGTGTGGATGTTAGATGCTGAAGATTTGGCTGATATTGAAGATACTGTGGAATGGTTGTACAGGAACAGCTTTAAGAACTATGTCATCACTCCCAGAATCTTCTGCAATGTGTAGAAAAGCAGCAAGGACTAGATTGCCTAGGGGAAAAGACTTAATTTACTTTGGGAGTGAAAAATCTGATCAAGAGACTGGACGTGTACTTCTGTTTCTCAGTGGAGGGCTGCCTTATTTGGAGCACCTTAATCTCTCTGGTTGTCTTACTATAACTGGTGCAGGCCTGCAGGATTTGGTTTCAGCATGTCCTTCTCTGAATGATGAATACTTTTACTACTGTGACAACATTAACGGTCCTCATGCTGATACCGCCAGTGGATGCCAGAATTTGCAGTGTGGTTTTCGAGCCTGCTGCCGCTCTGGCGAATGACCCTTGACTTCTGATCTTTGTCTACTTCATTTAGCTGAGCAGGCTTTCTTTCATGCACTTTACTCATAGCACATTTCTTGTGTTAACCATCCCTTTTTGAGCGTGACTTGTTTTGGCCCCATTTCTTACAACTTCAGAAATCTTAATTTACCAGTGAATTGTAATGTTGTTTCTCTTGCAAATTATACTTTTGGTTTAGAAAGGGATTAGGTCTTTTCAAAAGGGTGAGAACAGTCTTACATTTTTCTTTTAAATGAAATGCTTTAAAGAATGTTGGTAATGCCATGTCATTTAAAGTATTTCATAGATAATTTTGAGTTTTAAAGTCCATGGAGGTGATTGGTTCTCTTTACACATTAACACTGTACCAAGCTTTGCAGATCTTTTCCGACACACATGTCTGAAGACTTATTTTCAAAGACAGCACATTTTTGGAAACTAATCTCTTTTCCGTAATATTTCCTTTATTTCAATGATTCTCAGAAGGCCAATTCAAACAAACCCACATTTAAGGTTCTTTAGGATTATAGAATAAATTGGCTTCTGAGTGTTAGCTCAGTGAGTAGGAAAGCACCAATCGATATTTGTTTCCTTTAGGGATACTTTGTTCTCACCACTGTCCCTATGTCATCAAATTTGGGAGAGATTTTTTAAAATACCACAATCATTTGAAGAAATGTATAAATAAAATCTACTTTGAGGACTTTACC AAGTAA

[0363] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariation of the described method and system of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in molecular biology or related fields are intended to bewithin the scope of the following claims.

1 60 1 42 PRT Homo sapiens 1 Leu Pro Ala Arg Gly Leu Asp His Ile Ala GluAsn Ile Leu Ser Tyr 1 5 10 15 Leu Asp Ala Lys Ser Leu Cys Ala Ala GluLeu Val Cys Lys Glu Trp 20 25 30 Tyr Arg Val Thr Ser Asp Gly Met Leu Trp35 40 2 126 DNA Homo sapiens 2 ctgccagctc ggggattgga tcatattgctgagaacattc tgtcatacct ggatgccaaa 60 tcactatgtg ctgctgaact tgtgtgcaaggaatggtacc gagtgacctc tgatggcatg 120 ctgtgg 126 3 38 PRT Homo sapiens 3Leu Pro Lys Glu Leu Leu Leu Arg Ile Phe Ser Phe Leu Asp Ile Val 1 5 1015 Thr Leu Cys Arg Cys Ala Gln Ile Ser Lys Ala Trp Asn Ile Leu Ala 20 2530 Leu Asp Gly Ser Asn Trp 35 4 114 DNA Homo sapiens 4 ttacccaaagaacttctgtt aagaatattt tccttcttgg atatagtaac tttgtgccga 60 tgtgcacagatttccaaggc ttggaacatc ttagccctgg atggaagcaa ctgg 114 5 38 PRT Homosapiens 5 Leu Pro Tyr Glu Leu Ile Gln Leu Ile Leu Asn His Leu Thr LeuPro 1 5 10 15 Asp Leu Cys Arg Leu Ala Gln Thr Cys Lys Leu Leu Ser GlnHis Cys 20 25 30 Cys Asp Pro Leu Gln Tyr 35 6 71 DNA Homo sapiens 6ctaccttatg agcttattca gctgattctg aatcatctta cactaccaga cctgtgtaga 60ttagcacaga c 71 7 38 PRT Mus musculus 7 Leu Pro Tyr Glu Leu Ile Gln LeuIle Leu Asn His Leu Ser Leu Pro 1 5 10 15 Asp Leu Cys Arg Leu Ala GlnThr Cys Arg Leu Leu His Gln His Cys 20 25 30 Cys Asp Pro Leu Gln Tyr 358 114 DNA Mus musculus 8 ctaccatatg agctcattca actgattctg aatcatctttcactaccaga cctgtgtaga 60 ttagcccaga cttgcaggct tctccaccag cattgctgtgatcctctgca atat 114 9 38 PRT Homo sapiens 9 Leu Pro Thr Asp Pro Leu LeuLeu Ile Leu Ser Phe Leu Asp Tyr Arg 1 5 10 15 Asp Leu Ile Asn Cys CysTyr Val Ser Arg Arg Leu Ser Gln Leu Ser 20 25 30 Ser His Asp Pro Leu Trp35 10 114 DNA Homo sapiens 10 ctgcccaccg atcccctgct cctcatcttatcctttttgg actatcggga tctaatcaac 60 tgttgttatg tcagtcgaag acttagccagctatcaagtc atgatccgct gtgg 114 11 38 PRT Mus musculus 11 Leu Pro Thr AspPro Leu Leu Leu Ile Val Ser Phe Val Asp Tyr Arg 1 5 10 15 Asp Leu IleAsn Cys Cys Tyr Val Ser Arg Ser Val Ser Gln Leu Ser 20 25 30 Thr His AspPro Leu Trp 35 12 114 DNA Mus musculus 12 ctacccaccg accctctgctcctcatagta tccttcgtgg actacaggga cctaatcaat 60 tgttgctatg ttagtcgaagcgttagccag ctatcaactc atgatccact gtgg 114 13 38 PRT Homo sapiens 13 LeuPro Pro Glu Val Met Leu Ser Ile Phe Ser Tyr Leu Asn Pro Gln 1 5 10 15Glu Leu Cys Arg Cys Ser Gln Val Ser Met Lys Trp Ser Gln Leu Thr 20 25 30Lys Thr Gly Ser Leu Trp 35 14 113 DNA Homo sapiens 14 cttcctcctgaggtaatgct gtcaattttc agctatctta atcctcaaga gttattcgat 60 gcagtcaagtaagcatgaaa tggtctcagc tgacaaaaac gggatcgctt tgg 113 15 38 PRT Musmusculus 15 Leu Pro Pro Glu Val Met Leu Ser Ile Phe Ser Tyr Leu Asn ProGln 1 5 10 15 Glu Leu Cys Arg Cys Ser Gln Val Ser Thr Lys Trp Ser GlnLeu Ala 20 25 30 Lys Thr Gly Ser Leu Trp 35 16 114 DNA Mus musculus 16cttcctcctg aggtaatgct gtccattttc agttacctta atcctcaaga attgtgtcgg 60tgtagtcaag tcagtactaa gtggtctcag ctggcaaaaa caggatcttt gtgg 114 17 41PRT Homo sapiens 17 Leu Pro Leu Glu Met Leu Thr Tyr Ile Leu Ser Phe LeuPro Leu Ser 1 5 10 15 Asp Gln Lys Glu Ala Ser Leu Val Ser Trp Ala TrpTyr Arg Ala Ala 20 25 30 Gln Asn Ala Leu Arg Glu Arg Leu Trp 35 40 18123 DNA Homo sapiens 18 ctgcccctgg agatgctcac atatattctg agcttcctgcctctgtcaga tcagaaagag 60 gcctccctcg tgagttgggc ttggtaccgt gctgcccagaatgcccttcg ggagaggctg 120 tgg 123 19 35 PRT Homo sapiens 19 Leu Pro ProGlu Leu Ser Phe Thr Ile Leu Ser Tyr Leu Asn Ala Thr 1 5 10 15 Asp LeuCys Leu Ala Ser Cys Val Trp Gln Asp Leu Ala Asn Asp Glu 20 25 30 Leu LeuTrp 35 20 105 DNA Homo sapiens 20 ttgcctcctg agctaagctt taccatcttgtcctacctga atgcaactga cctttgcttg 60 gcttcatgtg tttggcagga ccttgcgaatgatgaacttc tctgg 105 21 35 PRT Mus musculus 21 Leu Pro Pro Glu Leu SerPhe Thr Ile Leu Ser Tyr Leu Asn Ala Ile 1 5 10 15 Asp Leu Cys Leu AlaSer Cys Val Trp Gln Asp Leu Ala Asn Asp Glu 20 25 30 Leu Leu Trp 35 22105 DNA Mus musculus 22 ctgcctcctg agctgagcct caccatccta tcccacctggatgcaactga cctttgccta 60 gcttcctgtg gttggcaaga actcgctaat gatgaacttctctgg 105 23 38 PRT Mus musculus 23 Leu Pro Arg Val Leu Ser Val Tyr IlePhe Ser Phe Leu Asp Pro Arg 1 5 10 15 Ser Leu Cys Arg Cys Ala Gln ValSer Trp Tyr Trp Lys Ser Leu Ala 20 25 30 Glu Leu Asp Gln Leu Trp 35 24114 DNA Mus musculus 24 cttccaaggg tgttatctgt ctacatcttt tccttcctggatccccggag tctttgccgt 60 tgtgcacagg tgagctggta ctggaagagc ttggctgagttggaccagct ctgg 114 25 38 PRT Homo sapiens 25 Leu Pro Ile Asp Val GlnLeu Tyr Ile Leu Ser Phe Leu Ser Pro His 1 5 10 15 Asp Leu Cys Gln LeuGly Ser Thr Asn His Tyr Trp Asn Glu Thr Val 20 25 30 Arg His Pro Ile LeuTrp 35 26 114 DNA Homo sapiens 26 ctgccgattg atgtacagct atatattttgtcctttcttt cacctcatga tctgtgtcag 60 ttgggaagta caaatcatta ttggaatgaaactgtaagac atccaattct ttgg 114 27 40 PRT Homo sapiens 27 Leu Pro Leu GluLeu Trp Arg Met Ile Leu Ala Tyr Leu His Leu Pro 1 5 10 15 Asp Leu GlyArg Cys Ser Leu Val Cys Arg Ala Trp Tyr Glu Leu Ile 20 25 30 Leu Ser LeuAsp Ser Thr Arg Trp 35 40 28 120 DNA Homo sapiens 28 ctccccttggagctgtggcg catgatctta gcctacttgc accttcccga cctgggccgc 60 tgcagcctggtatgcagggc ctggtatgaa ctgatcctca gtctcgacag cacccgctgg 120 29 33 PRT Musmusculus 29 Leu Pro Ala Glu Ile Thr Phe Lys Ile Phe Ser Gln Leu Asp IleArg 1 5 10 15 Ser Leu Cys Arg Ala Ser Leu Thr Cys Arg Ser Trp Asn AspPhe Lys 20 25 30 Ser 30 90 DNA Mus musculus 30 ctgcctgcag aaatcacttttaaaattttc agtcagctgg acattcggag tctgtgcagg 60 gcttcattga catgcaggagctggaatgac 90 31 38 PRT Mus musculus 31 Leu Pro Leu Leu Gln Gln Pro LeuLeu Cys Ser Val Ala His Pro Ile 1 5 10 15 Ala Ser Phe Thr Met Leu SerTyr Leu Thr Gly Lys Glu Ala Ala His 20 25 30 Leu Ser Val Glu Leu Trp 3532 114 DNA Mus musculus 32 ctgccattac tgcagcagcc acttctgtgt tctgtggctcatcccatcgc cagcttcacc 60 atgctgtcat acctcacggg aaaggaggcc gctcatctgtcagtggagtt gtgg 114 33 38 PRT Mus musculus 33 Leu Pro Asp Ser Leu ValTyr Gln Ile Phe Leu Ser Leu Gly Pro Ala 1 5 10 15 Asp Val Leu Ala AlaGly Leu Val Cys Arg Gln Trp Gln Ala Val Ser 20 25 30 Arg Asp Glu Phe LeuTrp 35 34 114 DNA Mus musculus 34 ctccccgaca gccttgtcta ccagatcttcctgagtttgg gccctgcaga tgtgctggct 60 gctgggctgg tatgccgcca atggcaggctgtgtcccggg atgagttctt atgg 114 35 31 PRT Mus musculus 35 Leu Pro Glu GluVal Leu Ala Leu Ile Phe Arg Asp Leu Pro Leu Arg 1 5 10 15 Asp Leu AlaVal Ala Thr Arg Val Cys Arg Ala Trp Ala Ala Ala 20 25 30 36 93 DNA Musmusculus 36 ctgccagagg aagtgttggc gctcatcttc cgtgacctgc ctctcagggaccttgctgta 60 gccaccagag tctgcagggc ctgggcggcg gct 93 37 38 PRT Musmusculus 37 Leu Pro Ser Val Pro Met Met Glu Ile Leu Ser Tyr Leu Asp AlaTyr 1 5 10 15 Ser Leu Leu Gln Ala Ala Gln Val Asn Lys Asn Trp Asn GluLeu Ala 20 25 30 Ser Ser Asp Val Leu Trp 35 38 114 DNA Mus musculus 38ttacctagtg tgccgatgat ggaaatcctc tcctatctgg atgcctacag tttgctacag 60gctgcccaag tgaacaagaa ctggaatgaa cttgcaagca gtgatgtcct gtgg 114 39 38PRT Mus musculus 39 Met Pro Ser Glu Ile Leu Val Lys Ile Leu Ser Tyr LeuAsp Ala Val 1 5 10 15 Thr Leu Val Cys Ile Gly Cys Val Ser Arg Arg PheTyr His Leu Ala 20 25 30 Asp Asp Asn Leu Ile Trp 35 40 114 DNA Musmusculus 40 atgccatcgg aaatcttggt gaagatactt tcttacttgg atgcggtgaccttggtgtgc 60 attggatgtg tgagcagacg cttttatcat ttggctgatg acaatcttatttgg 114 41 43 PRT Homo sapiens 41 Leu Pro Met Glu Val Leu Met Tyr IlePhe Arg Trp Val Val Ser Ser 1 5 10 15 Asp Leu Asp Leu Arg Ser Leu GluGln Leu Ser Leu Val Cys Arg Gly 20 25 30 Phe Tyr Ile Cys Ala Arg Asp ProGlu Ile Trp 35 40 42 129 DNA Homo sapiens 42 ctgccaatgg aggtcctgatgtacatcttc cgatgggtgg tgtctagtga cttggacctc 60 agatcattgg agcagttgtcgctggtgtgc agagggttct acatctgtgc cagagaccct 120 gaaatatgg 129 43 18 PRTMus musculus 43 Leu Ser Leu Val Cys Arg Gly Phe Tyr Ile Cys Ala Arg AspPro Glu 1 5 10 15 Ile Trp 44 81 DNA Mus musculus 44 gacttggacctcagatcgtt agagcagttg tcactggtgt gcagaggatt ctatatctgt 60 gccagagaccctgaaatctg g 81 45 31 PRT Homo sapiens 45 Leu Pro Tyr Glu Leu Ala IleAsn Ile Phe Xaa Tyr Leu Asp Arg Lys 1 5 10 15 Glu Leu Gly Arg Cys AlaGln Val Ser Lys Thr Trp Glu Gly Asp 20 25 30 46 93 DNA Homo sapiens 46ctgccttacg aattggcaat caatatattt agtatctgga caggaaagaa ctaggaagat 60gtgcacaggt gagcaagacg tgggaaggtg att 93 47 38 PRT Homo sapiens 47 LeuPro Leu Glu Leu Lys Leu Arg Ile Phe Arg Leu Leu Asp Val Arg 1 5 10 15Ser Val Leu Ser Leu Ser Ala Val Cys Arg Asp Leu Phe Thr Ala Ser 20 25 30Asn Asp Pro Leu Leu Trp 35 48 114 DNA Homo sapiens 48 ctcccattggaactgaaact acggatcttc cgacttctgg atgttcgttc cgtcttgtct 60 ttgtctgcggtttgtcgtga cctctttact gcttcaaatg acccactcct gtgg 114 49 38 PRT Musmusculus 49 Leu Pro Leu Glu Leu Lys Leu Arg Ile Phe Arg Leu Leu Asp ValHis 1 5 10 15 Ser Val Leu Ala Leu Ser Ala Val Cys His Asp Leu Leu IleAla Ser 20 25 30 Asn Asp Pro Leu Leu Trp 35 50 114 DNA Mus musculus 50cttccactgg agctgaaact acgcatcttc cgacttttgg atgttcattc tgtcctggcc 60ctgtctgcag tctgtcatga cctcctcatt gcgtcaaatg acccactgct gtgg 114 51 456PRT Homo sapiens 51 Ser Ala Met Val Phe Ser Asn Asn Asp Glu Gly Leu IleAsn Lys Lys 1 5 10 15 Leu Pro Lys Glu Leu Leu Leu Arg Ile Phe Ser PheLeu Asp Ile Val 20 25 30 Thr Leu Cys Arg Cys Ala Gln Ile Ser Lys Ala TrpAsn Ile Leu Ala 35 40 45 Leu Asp Gly Ser Asn Trp Gln Arg Ile Asp Leu PheAsn Phe Gln Ile 50 55 60 Asp Val Glu Gly Arg Val Val Glu Asn Ile Ser LysArg Cys Gly Gly 65 70 75 80 Phe Leu Arg Lys Leu Ser Leu Arg Gly Cys IleGly Val Gly Asp Ser 85 90 95 Ser Leu Lys Thr Phe Ala Gln Asn Cys Arg AsnIle Glu His Leu Asn 100 105 110 Leu Asn Gly Cys Thr Lys Ile Thr Asp SerThr Cys Tyr Ser Leu Ser 115 120 125 Arg Phe Cys Ser Lys Leu Lys His LeuAsp Leu Thr Ser Cys Val Ser 130 135 140 Ile Thr Asn Ser Ser Leu Lys GlyIle Ser Glu Gly Cys Arg Asn Leu 145 150 155 160 Glu Tyr Leu Asn Leu SerTrp Cys Asp Gln Ile Thr Lys Asp Gly Ile 165 170 175 Glu Ala Leu Val ArgGly Cys Arg Gly Leu Lys Ala Leu Leu Leu Arg 180 185 190 Gly Cys Thr GlnLeu Glu Asp Glu Ala Leu Lys His Ile Gln Asn Tyr 195 200 205 Cys His GluLeu Val Ser Leu Asn Leu Gln Ser Cys Ser Arg Ile Thr 210 215 220 Asp GluGly Val Val Gln Ile Cys Arg Gly Cys His Arg Leu Gln Ala 225 230 235 240Leu Cys Leu Ser Gly Cys Ser Asn Leu Thr Asp Ala Ser Leu Thr Ala 245 250255 Leu Gly Leu Asn Cys Pro Arg Leu Gln Ile Leu Glu Ala Ala Arg Cys 260265 270 Ser His Leu Thr Asp Ala Gly Phe Thr Leu Leu Ala Arg Asn Cys His275 280 285 Glu Leu Glu Lys Met Asp Leu Glu Glu Cys Ile Leu Ile Thr AspSer 290 295 300 Thr Leu Ile Gln Leu Ser Ile His Cys Pro Lys Leu Gln AlaLeu Ser 305 310 315 320 Leu Ser His Cys Glu Leu Ile Thr Asp Asp Gly IleLeu His Leu Ser 325 330 335 Asn Ser Thr Cys Gly His Glu Arg Leu Arg ValLeu Glu Leu Asp Asn 340 345 350 Cys Leu Leu Ile Thr Asp Val Ala Leu GluHis Leu Glu Thr Ala Glu 355 360 365 Ala Trp Ser Ala Ser Ser Cys Thr ThrAla Ser Arg Leu Pro Val Gln 370 375 380 Ala Ser Ser Gly Cys Gly Leu SerSer Leu Met Ser Lys Ser Thr Pro 385 390 395 400 Thr Leu Leu Pro Ser ProHis Arg Gln Gln Trp Gln Glu Val Asp Ser 405 410 415 Asp Cys Ala Gly AlaVal Ser Phe Ser Asp Ser Ser Cys Leu Gly Pro 420 425 430 Arg Gly Asp GluAla Ser Phe Pro Leu Glu Asp Leu Ser Leu Pro Asp 435 440 445 Arg Leu HisHis His Pro Ile Cys 450 455 52 1230 DNA Homo sapiens 52 ttcggccatggttttctcaa acaatgatga aggccttatt aacaaaaagt tacccaaaga 60 acttctgttaagaatatttt ccttcttgga tatagtaact ttgtgccgat gtgcacagat 120 ttccaaggcttggaacatct tagccctgga tggaagcaac tggcaaagaa tagatctttt 180 taactttcaaatagatgtag agggtcgagt ggtggaaaat atctcgaagc gatgcggtgg 240 attcctgaggaagctcagct tgcgaggctg cattggtgtt ggggattcct ccttgaagac 300 ctttgcacagaactgccgaa acattgaaca tttgaacctc aatggatgca caaaaatcac 360 tgacagcacgtgttatagcc ttagcagatt ctgttccaag ctgaaacatc tggatctgac 420 ctcctgtgtgtctattacaa acagctcctt gaaggggatc agtgagggct gccgaaacct 480 ggagtacctgaacctctctt ggtgtgatca gatcacgaag gatggcatcg aggcactggt 540 gcgaggttgtcgaggcctga aagccctgct cctgaggggc tgcacacagt tagaagatga 600 agctctgaaacacattcaga attactgcca tgagcttgtg agcctcaact tgcagtcctg 660 ctcacgtatcacggatgaag gtgtggtgca gatatgcagg ggctgtcacc ggctacaggc 720 tctctgcctttcgggttgca gcaacctcac agatgcctct cttacagccc tgggtttgaa 780 ctgtccgcgactgcaaattt tggaggctgc ccgatgctcc catttgactg acgcaggttt 840 tacacttttagctcggaatt gccacgaatt ggagaagatg gatcttgaag aatgcatcct 900 gataaccgacagcacactca tccagctctc cattcactgt cctaaactgc aagccctgag 960 cctgtcccactgtgaactca tcacagatga tgggatcctg cacctgagca acagtacctg 1020 tggccatgagaggctgcggg tactggagtt ggacaactgc ctcctcatca ctgatgtggc 1080 cctggaacacctagaaactg ccgaggcctg gagcgcctcg agctgtacga ctgccagcag 1140 gttacccgtgcaggcatcaa gcggatgcgg gctcagctcc ctcatgtcaa agtccacgcc 1200 tactttgctcccgtcacccc accgacagca 1230 53 380 PRT Homo sapiens 53 Arg Pro Arg PheGly Thr Ser Asp Ile Glu Asp Asp Ala Tyr Ala Glu 1 5 10 15 Lys Asp GlyCys Gly Met Asp Ser Leu Asn Lys Lys Phe Ser Ser Ala 20 25 30 Val Leu GlyGlu Gly Pro Asn Asn Gly Tyr Phe Asp Lys Leu Pro Tyr 35 40 45 Glu Leu IleGln Leu Ile Leu Asn His Leu Thr Leu Pro Asp Leu Cys 50 55 60 Arg Leu AlaGln Thr Cys Lys Leu Leu Ser Gln His Cys Cys Asp Pro 65 70 75 80 Leu GlnTyr Ile His Leu Asn Leu Gln Pro Tyr Trp Ala Lys Leu Asp 85 90 95 Asp ThrSer Leu Glu Phe Leu Gln Ser Arg Cys Thr Leu Val Gln Trp 100 105 110 LeuAsn Leu Ser Trp Thr Gly Asn Arg Gly Phe Ile Ser Val Ala Gly 115 120 125Phe Ser Arg Phe Leu Lys Val Cys Gly Ser Glu Leu Val Arg Leu Glu 130 135140 Leu Ser Cys Ser His Phe Leu Asn Glu Thr Cys Leu Glu Val Ile Ser 145150 155 160 Glu Met Cys Pro Asn Leu Gln Ala Leu Asn Leu Ser Ser Cys AspLys 165 170 175 Leu Pro Pro Gln Ala Phe Asn His Ile Ala Lys Leu Cys SerLeu Lys 180 185 190 Arg Leu Val Leu Tyr Arg Thr Lys Val Glu Gln Thr AlaLeu Leu Ser 195 200 205 Ile Leu Asn Phe Cys Ser Glu Leu Gln His Leu SerLeu Gly Ser Cys 210 215 220 Val Met Ile Glu Asp Tyr Asp Val Ile Ala SerMet Ile Gly Ala Lys 225 230 235 240 Cys Lys Lys Leu Arg Thr Leu Asp LeuTrp Arg Cys Lys Asn Ile Thr 245 250 255 Glu Asn Gly Ile Ala Glu Leu AlaSer Gly Cys Pro Leu Leu Glu Glu 260 265 270 Leu Asp Leu Gly Trp Cys ProThr Leu Gln Ser Ser Thr Gly Cys Phe 275 280 285 Thr Arg Leu Ala His GlnLeu Pro Asn Leu Gln Lys Leu Phe Leu Thr 290 295 300 Ala Asn Arg Ser ValCys Asp Thr Asp Ile Asp Glu Leu Ala Cys Asn 305 310 315 320 Cys Thr ArgLeu Gln Gln Leu Asp Ile Leu Gly Lys Val Thr Ile Tyr 325 330 335 Lys PheVal Leu Asn Val Cys Phe Leu Asp Arg Lys Ala Asn Leu Arg 340 345 350 LeuPhe Val Arg Lys Lys Lys Ile Phe Gly Tyr Asn Lys Asn Phe Ile 355 360 365Leu Ile Arg Trp Leu Gly Leu Ile Gly Asn Ala Arg 370 375 380 54 1380 DNAHomo sapiens 54 aggccaagat tcggcacgag tgatatagaa gatgatgcct atgcagaaaaggatggttgt 60 ggaatggaca gtcttaacaa aaagtttagc agtgctgtcc tcggggaagggccaaataat 120 gggtattttg ataaactacc ttatgagctt attcagctga ttctgaatcatcttacacta 180 ccagacctgt gtagattagc acagacttgc aaactactga gccagcattgctgtgatcct 240 ctgcaataca tccacctcaa tctgcaacca tactgggcaa aactagatgacacttctctg 300 gaatttctac agtctcgctg cactcttgtc cagtggctta atttatcttggactggcaat 360 agaggcttca tctctgttgc aggatttagc aggtttctga aggtttgtggatccgaatta 420 gtacgccttg aattgtcttg cagccacttt cttaatgaaa cttgcttagaagttatttct 480 gagatgtgtc caaatctaca ggccttaaat ctctcctcct gtgataagctaccacctcaa 540 gctttcaacc acattgccaa gttatgcagc cttaaacgac ttgttctctatcgaacaaaa 600 gtagagcaaa cagcactgct cagcattttg aacttctgtt cagagcttcagcacctcagt 660 ttaggcagtt gtgtcatgat tgaagactat gatgtgatag ctagcatgataggagccaag 720 tgtaaaaaac tccggaccct ggatctgtgg agatgtaaga atattactgagaatggaata 780 gcagaactgg cttctgggtg tccactactg gaggagcttg accttggctggtgcccaact 840 ctgcagagca gcaccgggtg cttcaccaga ctggcacacc agctcccaaacttgcaaaaa 900 ctctttctta cagctaatag atctgtgtgt gacacagaca ttgatgaattggcatgtaat 960 tgtaccaggt tacagcagct ggacatatta ggtaaggtta caatatataaatttgtttta 1020 aatgtctgtt tccttgacag aaaagccaat ctcagacttt ttgttaggaaaaagaaaatt 1080 tttggataca ataaaaattt tatcctgata agatggcttg gtttgataggaaatgccaga 1140 tagatcagtt aatataggga ataattatat atgtacttta ataaaatagtgaggacaata 1200 acaattttat agttgaactg taaaaaacta taaccattaa ttcttggtctacttgtaaga 1260 gtgagaattt acatgagctg cgctctctat ttttattaag gagagaagaaattaattcat 1320 ttgtataatg aattcaagct agtttttttt aagtttctta attaagcggccgcaagctta 1380 55 519 PRT Homo sapiens 55 Met Val Ile Met Leu Glx GluArg Gln Lys Phe Phe Lys Tyr Ser Val 1 5 10 15 Asp Glu Lys Ser Asp LysGlu Ala Glu Val Ser Glu His Ser Thr Gly 20 25 30 Ile Thr His Leu Pro ProGlu Val Met Leu Ser Ile Phe Ser Tyr Leu 35 40 45 Asn Pro Gln Glu Leu CysArg Cys Ser Gln Val Ser Met Lys Trp Ser 50 55 60 Gln Leu Thr Lys Thr GlySer Leu Trp Lys His Leu Tyr Pro Val His 65 70 75 80 Trp Ala Arg Gly AspTrp Tyr Ser Gly Pro Ala Thr Glu Leu Asp Thr 85 90 95 Glu Pro Asp Asp GluTrp Val Lys Asn Arg Lys Asp Glu Ser Arg Ala 100 105 110 Phe His Glu TrpAsp Glu Asp Ala Asp Ile Asp Glu Ser Glu Glu Ser 115 120 125 Ala Glu GluSer Ile Ala Ile Ser Ile Ala Gln Met Glu Lys Arg Leu 130 135 140 Leu HisGly Leu Ile His Asn Val Leu Pro Tyr Val Gly Thr Ser Val 145 150 155 160Lys Thr Leu Val Leu Ala Tyr Ser Ser Ala Val Ser Ser Lys Met Val 165 170175 Arg Gln Ile Leu Glu Leu Cys Pro Asn Leu Glu His Leu Asp Leu Thr 180185 190 Gln Thr Asp Ile Ser Asp Ser Ala Phe Asp Ser Trp Ser Trp Leu Gly195 200 205 Cys Cys Gln Ser Leu Arg His Leu Asp Leu Ser Gly Cys Glu LysIle 210 215 220 Thr Asp Val Ala Leu Glu Lys Ile Ser Arg Ala Leu Gly IleLeu Thr 225 230 235 240 Ser His Gln Ser Gly Phe Leu Lys Thr Ser Thr SerLys Ile Thr Ser 245 250 255 Thr Ala Trp Lys Asn Lys Asp Ile Thr Met GlnSer Thr Lys Gln Tyr 260 265 270 Ala Cys Leu His Asp Leu Thr Asn Lys GlyIle Gly Glu Glu Ile Asp 275 280 285 Asn Glu His Pro Trp Thr Lys Pro ValSer Ser Glu Asn Phe Thr Ser 290 295 300 Pro Tyr Val Trp Met Leu Asp AlaGlu Asp Leu Ala Asp Ile Glu Asp 305 310 315 320 Thr Val Glu Trp Arg HisArg Asn Val Glu Ser Leu Cys Val Met Glu 325 330 335 Thr Ala Ser Asn PheSer Cys Ser Thr Ser Gly Cys Phe Ser Lys Asp 340 345 350 Ile Val Gly LeuArg Thr Ser Val Cys Trp Gln Gln His Cys Ala Ser 355 360 365 Pro Ala PheAla Tyr Cys Gly His Ser Phe Cys Cys Thr Gly Thr Ala 370 375 380 Leu ArgThr Met Ser Ser Leu Pro Glu Ser Ser Ala Met Cys Arg Lys 385 390 395 400Ala Ala Arg Thr Arg Leu Pro Arg Gly Lys Asp Leu Ile Tyr Phe Gly 405 410415 Ser Glu Lys Ser Asp Gln Glu Thr Gly Arg Val Leu Leu Phe Leu Ser 420425 430 Leu Ser Gly Cys Tyr Gln Ile Thr Asp His Gly Leu Arg Val Leu Thr435 440 445 Leu Gly Gly Gly Leu Pro Tyr Leu Glu His Leu Asn Leu Ser GlyCys 450 455 460 Leu Thr Ile Thr Gly Ala Gly Leu Gln Asp Leu Val Ser AlaCys Pro 465 470 475 480 Ser Leu Asn Asp Glu Tyr Phe Tyr Tyr Cys Asp AsnIle Asn Gly Pro 485 490 495 His Ala Asp Thr Ala Ser Gly Cys Gln Asn LeuGln Cys Gly Phe Arg 500 505 510 Ala Cys Cys Arg Ser Gly Glu 515 56 2276DNA Homo sapiens 56 atggtaatca tgctgtaaga gcgacagaaa ttttttaaatattccgtgga tgaaaagtca 60 gataaagaag cagaagtgtc agaacactcc acaggtataacccatcttcc tcctgaggta 120 atgctgtcaa ttttcagcta tcttaatcct caagagttatgtcgatgcag tcaagtaagc 180 atgaaatggt ctcagctgac aaaaacggga tcgctttggaaacatcttta ccctgttcat 240 tgggccagag gtgactggta tagtggtccc gcaactgaacttgatactga acctgatgat 300 gaatgggtga aaaataggaa agatgaaagt cgtgcttttcatgagtggga tgaagatgct 360 gacattgatg aatctgaaga gtctgcggag gaatcaattgctatcagcat tgcacaaatg 420 gaaaaacgtt tactccatgg cttaattcat aacgttctaccatatgttgg tacttctgta 480 aaaaccttag tattagcata cagctctgca gtttccagcaaaatggttag gcagatttta 540 gagctttgtc ctaacctgga gcatctggat cttacccagactgacatttc agattctgca 600 tttgacagtt ggtcttggct tggttgctgc cagagtcttcggcatcttga tctgtctggt 660 tgtgagaaaa tcacagatgt ggccctagag aagatttccagagctcttgg aattctgaca 720 tctcatcaaa gtggcttttt gaaaacatct acaagcaaaattacttcaac tgcgtggaaa 780 aataaagaca ttaccatgca gtccaccaag cagtatgcctgtttgcacga tttaactaac 840 aagggcattg gagaagaaat agataatgaa cacccctggactaagcctgt ttcttctgag 900 aatttcactt ctccttatgt gtggatgtta gatgctgaagatttggctga tattgaagat 960 actgtggaat ggagacatag aaatgttgaa agtctttgtgtaatggaaac agcatccaac 1020 tttagttgtt ccacctctgg ttgttttagt aaggacattgttggactaag gactagtgtc 1080 tgttggcagc agcattgtgc ttctccagcc tttgcgtattgtggtcactc attttgttgt 1140 acaggaacag ctttaagaac tatgtcatca ctcccagaatcttctgcaat gtgtagaaaa 1200 gcagcaagga ctagattgcc taggggaaaa gacttaatttactttgggag tgaaaaatct 1260 gatcaagaga ctggacgtgt acttctgttt ctcagtttatctggatgtta tcagatcaca 1320 gaccatggtc tcagggtttt gactctggga ggagggctgccttatttgga gcaccttaat 1380 ctctctggtt gtcttactat aactggtgca ggcctgcaggatttggtttc agcatgtcct 1440 tctctgaatg atgaatactt ttactactgt gacaacattaacggtcctca tgctgatacc 1500 gccagtggat gccagaattt gcagtgtggt tttcgagcctgctgccgctc tggcgaatga 1560 cccttgactt ctgatctttg tctacttcat ttagctgagcaggctttctt tcatgcactt 1620 tactcatagc acatttcttg tgttaaccat ccctttttgagcgtgacttg ttttggcccc 1680 atttcttaca acttcagaaa tcttaattta ccagtgaattgtaatgttgt ttctcttgca 1740 aattatactt ttggtttaga aagggattag gtcttttcaaaagggtgaga acagtcttac 1800 atttttcttt taaatgaaat gctttaaaga atgttggtaatgccatgtca tttaaagtat 1860 ttcatagata attttgagtt ttaaagtcca tggaggtgattggttctctt tacacattaa 1920 cactgtacca agctttgcag atcttttccg acacacatgtctgaagactt attttcaaag 1980 acagcacatt tttggaaact aatctctttt ccgtaatatttcctttattt caatgattct 2040 cagaaggcca attcaaacaa acccacattt aaggttctttaggattatag aataaattgg 2100 cttctgagtg ttagctcagt gagtaggaaa gcaccaatcgatatttgttt cctttaggga 2160 tactttgttc tcaccactgt ccctatgtca tcaaatttgggagagatttt ttaaaatacc 2220 acaatcattt gaagaaatgt ataaataaaa tctactttgaggactttacc aagtaa 2276 57 39 PRT Homo sapiens 57 Leu Pro Leu Glu Leu SerPhe Tyr Leu Leu Lys Trp Leu Asp Pro Gln 1 5 10 15 Thr Leu Leu Thr CysCys Leu Val Ser Lys Gln Trp Asn Lys Val Ile 20 25 30 Ser Ala Cys Thr GluVal Trp 35 58 117 DNA Homo sapiens 58 cttcccctgg agctcagttt ttatttgttaaaatggctcg atcctcagac tttactcaca 60 tgctgcctcg tctctaaaca gtggaataaggtgataagtg cctgtacaga ggtgtgg 117 59 10 DNA Artificial SequenceDescription of Artificial Sequence Synthetic 59 aattcgcgcg 10 60 21 PRTArtificial Sequence Description of Artificial Sequence Synthetic 60 LysLys Glu Arg Leu Leu Asp Asp Arg His Asp Ser Gly Leu Asp Ser 1 5 10 15Met Lys Asp Glu Glu 20

What is claimed is:
 1. A method for the detection of one or more NF-κBregulatory factors comprising the steps of: a) providing a slimbprotein, and a sample suspected of containing one or more NF-κBregulatory factors; and b) exposing said slimb protein to said sampleunder conditions such that said slimb protein binds to said one or moreNF-κB regulatory factors to form a slimb/regulatory factor complex. 2.The method of claim 1, further comprising the step of detecting saidslimb/regulatory factor complex.
 3. The method of claim 1, furthercomprising the step of observing said slimb/regulatory factor complexfor degradation of said one or more NF-κB regulatory factors.
 4. Themethod of claim 1, further comprising the step of exposing said slimbprotein and one or more NF-κB regulatory factors to an F-box proteinantagonist.
 5. The method of claim 4, wherein said F-box proteinantagonist prevents the formation of said slimb/regulatory factorcomplex.
 6. A method for the detection of a slimb protein complex,comprising the steps of: a) providing a slimb protein and a samplesuspected of containing one or more proteins capable of forming acomplex with said slimb protein; and b) exposing said slimb protein tosaid one or more proteins capable of forming a complex with said slimbprotein under conditions such that said slimb protein binds to said oneor more proteins capable of forming a complex with said slimb protein toform a slimb protein complex.
 7. The method of claim 6, furthercomprising the step of detecting said slimb protein complex.
 8. Themethod of claim 6, wherein step b) further comprises exposing said slimbprotein and said one or more proteins capable of forming a complex withsaid slimb protein to an F-box protein antagonist.
 9. The method ofclaim 8, wherein said F-box protein antagonist prevents the formation ofsaid slimb protein complex.
 10. An isolated nucleotide sequencecomprising nucleotide sequence encoding at least one functionally activefragement of an F-box protein, wherein said sequence consists of a leasta portion of the sequence set forth in SEQ ID NOS: 54 and 56.